Mammalian cytokines; receptors; related reagents and methods

ABSTRACT

Nucleic acids encoding mammalian cytokine receptor, e.g., for cytokine IL-B50, purified proteins and fragments thereof. Antibodies, both polyclonal and monoclonal, are also provided. Methods of using the compositions for both diagnostic and therapeutic utilities are described.

RELATED REAGENTS AND METHODS

This application is a divisional of U.S. patent application Ser. No.10/927,228, filed Aug. 25, 2004, which is a divisional of U.S. patentapplication Ser. No. 10/008,566, filed Nov. 8, 2001, now U.S. Pat. No.6,890,734, which claims benefit of U.S. Provisional Patent ApplicationsNos. 60/298,268, filed Jun. 14, 2001, and 60/247,218, filed Nov. 10,2000, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compositions and methods for affectingmammalian physiology, including hematopoietic cell proliferation orimmune system function. In particular, it provides methods of usingnucleic acids, proteins, and antibodies which regulate developmentand/or the immune system; and provides functional details onligand-receptor pairing. Diagnostic and therapeutic uses of thesematerials are also disclosed.

BACKGROUND OF THE INVENTION

Recombinant DNA technology refers generally to techniques of integratinggenetic information from a donor source into vectors for subsequentprocessing, such as through introduction into a host, whereby thetransferred genetic information is copied and/or expressed in the newenvironment. Commonly, the genetic information exists in the form ofcomplementary DNA (cDNA) derived from messenger RNA (mRNA) coding for adesired protein product. The carrier is frequently a plasmid having thecapacity to incorporate cDNA for later replication in a host and, insome cases, actually to control expression of the cDNA and therebydirect synthesis of the encoded product in the host.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork”. Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of the immuneresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play critical roles in controllingthese cellular interactions. Thus, there is considerable interest in theisolation, characterization, and mechanisms of action of cell modulatoryfactors, an understanding of which will lead to significant advancementsin the diagnosis and therapy of numerous medical abnormalities, e.g.,immune system disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, and/ordifferentiation of pluripotent hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages which makeup a complex immune system. Proper and balanced interactions between thecellular components are necessary for a healthy immune response. Thedifferent cellular lineages often respond in a different manner whenlymphokines are administered in conjunction with other agents.

Cell lineages especially important to the immune response include twoclasses of lymphocytes: B-cells, which can produce and secreteimmunoglobulins (proteins with the capability of recognizing and bindingto foreign matter to effect its removal), and T-cells of various subsetsthat secrete lymphokines and induce or suppress the B-cells and variousother cells (including other T-cells) making up the immune network.These lymphocytes interact with many other cell types. Monocytes areprecursors of macrophages which, with dendritic cells, are functionallyimportant in their roles as processors and presenters of antigen, animportant step in initiation of an immune response.

IL-7 is a cell modulatory factor which affects hematopoietic cell growthand/or differentiation. See, e.g., Mire-Sluis and Thorpe (1998)Cytokines Academic Press, San Diego; Thomson (ed. 1998) The CytokineHandbook (3d ed.) Academic Press, San Diego; Metcalf and Nicola (1995)The Hematopoietic Colony Stimulating Factors Cambridge University Press;and Aggarwal and Gutterman (1991) Human Cytokines Blackwell.

Research to better understand and treat various immune disorders hasbeen hampered by the general inability to maintain cells of the immunesystem in vitro. Immunologists have discovered that culturing many ofthese cells can be accomplished through the use of T-cell and other cellsupernatants, which contain various growth factors, including many ofthe lymphokines.

From the foregoing, it is evident that the understanding of the signaltransduction pathways and identification of components in such pathwaysshould contribute to new therapies for a wide range of degenerative orabnormal conditions which directly or indirectly involve development,differentiation, or function, e.g., of the immune system and/orhematopoietic cells. Furhtermore, soluble regulatory molecules,including cytokines, are known to sometimes act outside the immunesystem with effects on physiology (leptin), morphogenesis, and tissueand skeletal remodeling (RANKL). Thus, the discovery and understandingof novel cytokine-like molecules and their receptors which enhance orpotentiate the beneficial activities of other lymphokines would behighly advantageous.

SUMMARY OF THE INVENTION

The present invention is based, in part, on the discovery of thereceptor complex for the cytokine ligand IL-B50. Moreover,identification of the components allows for the identification of celltypes and stages which express the necessary components to be responsiveto ligand. This provides insights and capacity to predict thephysiological and immunological role of the cytokine.

The present invention provides, e.g., methods of producing aligand:receptor complex, comprising contacting: a substantially pure orrecombinant mammalian IL-B50 with a receptor comprising the IL-7Rα orthe Rδ2 subunit; a mammalian IL-B50 with a receptor comprising asubstantially pure or recombinant IL-7Rα subunit; or a mammalian IL-B50with a receptor comprising a substantially pure or recombinant Rδ2subunit; which contacting thereby allows the complex to form. Inpreferred embodiments, the mammalian IL-B50 is primate IL-B50, such ashuman IL-B50; the complex formation results in signal transduction, STATactivation, or TARC expression; the receptor is on a cell; the receptorcomprises both IL-7Rα and Rδ2 subunit; the complex formation results ina physiological change in the cell expressing the receptor; thecontacting is in combination with a proliferative agent, cytokine, orchemokine; the contacting allows quantitative detection of the ligand;or receptor is on a hematopoietic cell, including a lymphoid lineagecell, a myeloid cell such as a monocyte, or dendritic cell.

Another method is provided for modulating physiology or development ofan IL-7Rα or Rδ2 expressing cell comprising contacting the cell to anexogenous agonist or antagonist of a mammalian IL-B50. Variousembodiments include those wherein: the antagonist is an antibody whichneutralizes the mammalian IL-B50, a mutein of the IL-B50; or an antibodywhich binds to IL-7Rα or Rδ2 or a complex of both; or the physiology isselected from proliferation, lymphoid lineage cell development, antigenpresentation, or production of inflammatory mediators, includingcytokines, chemokines, or adhesion molecules; or the cell is ahematopoietic cell. Other embodiments include those wherein: theantagonist is an antibody and the physiology is hematopoietic cellproliferation; the agonist is IL-B50 and the physiology is hematopoieticcell differentiation; the physiology is antigen presentation; or themodulating is blocking, and the physiology is lymphoid lineage cellproliferation.

Other embodiments provide methods of modulating a signal to a cellmediated by IL-B50 comprising contacting the cell to an administeredagonist or antagonist of IL-B50. These include those wherein themodulating is inhibiting, and the signal is a proliferation signal; theantagonist is a neutralizing antibody to IL-7Rα or the Rδ2 subunit or acomplex comprising the subunits; the agonist or antagonist isadministered in combination with another antagonist or agonist ofIL-B50; the agonist or antagonist is administered in combination with agrowth factor, cytokine, chemokine, or immune adjuvant; or thecontacting is with another anti-proliferative agent or treatment.

Other methods include those of selectively labeling a population ofcells, the method comprising contacting the cells with an antibody whichbinds: IL-7Rα; Rδ2; or a complex comprising one of the subunits; therebyresulting in the identification of cells expressing the subunit orcomplex. Certain embodiments include those wherein: the contactingresults in modulation of STAT activation; the labeling allowspurification of IL-7Rα or Rδ2 subunit expressing cells; or the labelingallows depletion of IL-7Rα or Rδ2 subunit expressing cells. Alsoprovided are populations of cells made by the methods, including thosewhich are prepared by Fluorescent Activated Cell Sorting.

The invention further provides methods of testing a compound for abilityto affect receptor-ligand interaction, the method comprising comparingthe interaction of a receptor complex comprising IL-7Rα and/or Rδ2subunit with IL-B50 in the presence and absence of the compound. Incertain embodiments, the compound is an antibody which binds one of:IL-7Rα; Rδ2 subunit; a receptor comprising IL-7R and/or Rδ2; or IL-B50.

Certain compositions are provided, e.g., an isolated or recombinantprotein complex comprising: at least 15 contiguous amino acid residuesof SEQ ID NO: 2 and at least 15 contiguous amino acid residues of SEQ IDNO: 4; at least two distinct segments of at least 8 contiguous aminoacid residues of SEQ ID NO: 2 and at least two distinct segments of atleast 8 contiguous amino acid residues of SEQ ID NO: 4; or at least onesegment at least 21 contiguous nucleotides of SEQ ID NO: 1 and at leastone segment at least 21 contiguous nucleotides of SEQ ID NO: 3. Inpreferred embodiments, one of the segments of SEQ ID NO: 2 is from theextracellular portion of the sequence; one of the segments of SEQ ID NO:4 is from the extracellular portion of the sequence; or the polypeptidecomprises the mature SEQ ID NO: 2 and the mature SEQ ID NO: 4 sequences.

Nucleic acid embodiments include an isolated or recombinantpolynucleotide encoding described components of the complex, wherein:the polynucleotide comprises a deoxyribonucleotide; the polynucleotidecomprises a ribonucleotide; or at least one of the segments is operablylinked to a promoter.

Antibodies are also provided which recognize epitopes presented by thecomplex, e.g., a binding compound comprising an antigen binding portionfrom an antibody which binds with selectivity to a polypeptidecomprising at least 12 contiguous amino acid residues of SEQ ID NO: 2and at least 12 contiguous amino acid residues of SEQ ID NO: 4.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1E show the nucleotide and amino acid sequences (see SEQ ID NO:1 and 2) of a primate, e.g., human, IL-7Rα; predicted signal cleavagesite indicated.

FIGS. 2A-2E show the nucleotide and amino acid sequences (see SEQ ID NO:3 and 4) of a primate, e.g., human, Rδ2; predicted signal cleavage siteindicated.

FIGS. 3A and 3B show the amino acid and nucleotide sequences,respectively, of primate, e.g., human, IL-B50; predicted signal cleavageposition indicated.

FIGS. 4A-4E show expression levels of hIL-7Rα (FIGS. 4A-4B), Rδ2(hTSLPR, FIGS. 4C-4D), and IL-B50 in various tissues and cell types.Expression levels were normalized and expressed as femtograms mRNA per50 ng total cDNA.

FIG. 5 shows the induction of TARC by IL-B50. Human CD11c+ DC werecultured in the absence or the presence of IL-B50 (50 ng/ml) and theproduction of TARC was determined in the culture supernatant by ELISA.

FIG. 6A depicts a culture of sorted CD11c+ DC after 24 h in mediumalone. DC form small and irregular clumps with a dark center, indicatingthe presence of dying cells.

FIG. 6B depicts a culture of sorted CD11c+ DC from the same donor as inFIG. 6A, treated with 15 ng/ml of IL-B50. DC form larger and roundclumps with fine dendrites visible at the periphery, indicating thematuration of the DC.

FIG. 7 shows the surface phenotype of CD11c+ DC after 24 h of culturewith and without (medium alone) IL-B50 and shows the upregulation ofHLA-DR, as well as the costimulatory molecules CD40, CD80 and CD86.Results shown are from one representative of four independentexperiments.

FIGS. 8A-8C show the surface phenotype of DC after treatment with mediumalone, IL-B50, CD40-ligand (CD40L), IL-7 and LPS. IL-B50 is more potentthan CD40-ligand and IL-7 in upregulating costimulatory molecules CD40and CD80.

FIG. 9 shows the results of a T cell proliferation assay using CD11c+ DCmatured for 24 h in medium or with IL-B50 (15 ng/ml) and cocultured with5×10⁴ allogenic CD4+CD45RA+ naïve T cells at increasing DC/T cellratios. Proliferation was assessed on day 6 by measuring [³H]thymidineincorporation. Each point represents the mean [³H]thymidineincorporation of triplicate co cultures. Vertical bars indicate the SD.DC alone (□) were used as a control and did not significantlyproliferate. Results shown are from one representative of the twoindependent experiments.

FIG. 10 shows the results of a similar experiment as described for FIG.9, using DC matured in medium, IL-B50, CD40-ligand (CD40L), IL-7 andLPS.

FIGS. 11A-11E show the production of various cytokines (expressed aspg/ml) by naïve CD4 T cells cocultured with DC matured in medium alone,IL-B50, CD40-ligand (CD40L), IL-7 and LPS. FIG. 11A shows the effect onthe production of IL-4; FIG. 11B shows the effect on the production ofIL-13; FIG. 11C shows the effect on the production of IFN-γ, FIG. 11Dshows the effect on the production of IL-10 and FIG. 11E shows theeffect on the production of TNF-α.

FIG. 12 shows the effect of DCs treated with medium alone, IL-B50, IL-7,and CD40-ligand on CD8 T cell expansion.

FIG. 13 compares expression of perforin by human naïve CD8 T cellsinduced by DCs treated with medium alone, IL-B50 or CD40-ligand.

FIGS. 14A-14C show the results of a comparison of IL-B50 with GM-CSF,IL-7, CD40-ligand (CD40L) and medium alone as a control, to stimulatehuman DCs to produce mRNA for various cytokines and chemokines. FIG. 14Ashows effects on IL-1α, IL-1β, IL-6, IL-12p40 and TNF-α. FIG. 14B showseffects on TARC, MDC and MIP3-β. FIG. 14C shows effects on MCP-1, MCP-4,Rantes and MIG.

FIG. 15 shows the effect of IL-B50 on the induction of IL12p75 protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Outline

-   I. General-   II. Activities-   III. Nucleic acids    -   A. encoding fragments, sequence, probes    -   B. mutations, chimeras, fusions    -   C. making nucleic acids    -   D. vectors, cells comprising-   IV. Proteins, Peptides    -   A. fragments, sequence, immunogens, antigens    -   B. muteins    -   C. agonists/antagonists, functional equivalents    -   D. making proteins-   V. Making nucleic acids, proteins-   VI. Antibodies    -   A. polyclonals    -   B. monoclonal, Kd    -   C. anti-idiotypic antibodies    -   D. hybridoma cell lines-   VII. Kits and Methods to quantify ligand/receptor    -   A. ELISA    -   B. assay mRNA encoding    -   C. qualitative/quantitative    -   D. kits-   VIII. Therapeutic compositions, methods    -   A. combination compositions    -   B. unit dose    -   C. administration-   IX. Receptors    I. General

Before the present compositions, formulations, and methods aredescribed, it is to be understood that this invention is not limited tothe particular methods, compositions, and cell lines described herein,as such methods, compositions, and cell lines may, of course, vary. Itis also to be understood that the terminology used herein is for thepurpose of describing particular embodiments.

As used herein, including the appended claims, singular forms of wordssuch as “a,” “an,” and “the” include their corresponding pluralreferents unless the context clearly dictates otherwise. Thus, e.g.,reference to “an organism” includes one or more different organisms,reference to “a cell” includes one or more of such cells, and referenceto “a method” includes reference to equivalent steps and methods knownto a person of ordinary skill in the art, and so forth.

Unless otherwise defined, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. Publications, patent applications,patents, and other references discussed above are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the invention isnot entitled to antedate any such disclosure by virtue of its priorinvention. Publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety including all figures and drawings.

The present invention is based on the discovery of the receptor subunitsfor the IL-B50 cytokine. This allows advantageous coproduction of thesubunits on vectors, production of fusion proteins, and antibodypreparations which recognize epitopes resulting from the interaction ofthe subunit components into a functional unit.

IL-7 biology is reasonably well described. See, e.g., Stoddart, et al.(2000) Immunol. Rev. 175:47-58; Puel and Leonard (2000) Curr. Opin.Immunol. 12:468-473; Akashi, et al. (2000) Curr. Opin. Immunol.12:144-150; Watanabe, et al. (1999) Immunol. Res. 20:251-259; Waldmann(2000) Ann. Oncol. 11 Suppl 1:101-106; Beverley and Grubeck-Loebenstein(2000) Vaccine 18:1721-1724; Aspinall and Andrew (2000) Vaccine18:1629-1637; Appasamy (1999) Cytokines Cell Mol. Ther. 5:25-39;Hofmeister, et al. (1999) Cytokine Growth Factor Rev. 10:41-60; Or, etal. (1998) Cytokines Cell Mol. Ther. 4:287-294; Akashi, et al. (1998)Immunol. Rev. 165:13-28; and Offner and Plum (1998) Leuk. Lymphoma30:87-99. Moreover, since the IL-7 receptor and the IL-B50 receptorshare one subunit, the signaling pathways and biology shouldsignificantly overlap. This is similar to the GM/IL-3/IL-5 family, whichis one of the first groups whose overlapping biologies were explained bythe sharing of receptor subunits.

Additionally, recognition of the receptor subunits provides theopportunity to determine cell types and developmental stages where thefunctional receptor components are coordinately expressed. This providesthe opportunity to determine what cell types are likely to respond toligand, and the resulting biological functions mediated by those cellsprovides suggestions as to the physiological effects mediated by theligand. This leads to better understanding of therapeutic uses of theligand or blocking ligand:receptor interaction and signaling.

Some of the standard methods applicable are described or referenced,e.g., in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, etal. (1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols 1-3,CSH Press, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and periodic supplements)Current Protocols in Molecular Biology, Greene/Wiley, New York; each ofwhich is incorporated herein by reference. See also U.S. Ser. No.09/130,972, which is incorporated herein by reference.

A complete nucleotide (SEQ ID NO: 1) and corresponding amino acidsequence (SEQ ID NO: 2) of a primate, e.g., human, IL-7Rα coding segmentis shown in FIG. 1; similarly for Rδ2 in FIG. 2. FIG. 3 providessequence of primate, e.g., human, IL-B50. See U.S. Ser. No. 09/399,492,which is incorporated herein by reference.

For the IL-7Rα subunit, notable predicted features include, e.g., CK2phosphorylation sites at about S24-E27, T47-E50, S187-D190, S214-E217,S255-D258, S282-D285, S355-D358, and S423-E426; peroxisomal motif atabout S133-L135; PKC phosphorylation sites at about T76-K78, T97-K99,S165-K167, S315-K317, S364-R366, and S373-K375; receptor cyt2 motif atabout G195-S201; cAMP kinase motif at about f342-S346; cAMP kinase motifat about R343-S346 or R343-L367; GSK3 motifs at about T129-S133,S201-Y205, T208-n212, S324-p330, T337-f341, S363-S367, and T413-S417; Nglycosylation sites at about N29-S31, N45-T47, N131-S133, N162-S164,N212-S214, N213-S215, N275-S277, N353-S355, and N392-T394; tyrosinekinase motif at about E14-Y18; cAMP PK sites at about K94-T97, K84-S87,R343-S346, Cas phos sites at about T208-K210, S215-E217, T262-E264,S331-D333, T337-E339, and S367-D369; cyt C me sites at about T76-F79,C98-I101, Q136-Y139, W244-R247, H259-T261, and C267-P270; histonemethylation sites at about F85-L88, Q172-L175, and P270-N273; myristolysites at about G322-S326, G352-A256, and G389-S392; Phos2 sites at aboutK184/S187, R343/S345, R343/S346, and R371/S373; and PKC phos sites atabout T76-K78, T97-K99, S165-K167, S315-K317, S364-R366, and S373-K375.

Regions of particular interest from the Rδ2 sequence include predictionsof, e.g., CK2 phosphorylation sites at about T115-D118, S120-D123,S132-D135, S140-E143, S242-D245, S284-E287, and T297-E300; peroxisomallocalization motifs at about S218-F220 and A272-L274; PKCphosphorylation sites at about S76-R78, S99-K101, and S300-R302; tyrphosphorylation sites at about R39/D43/Y46 and K166/E169/Y172; cAMPkinase sites at about i77-T81 and R78-T82; Ca++ kinase site at aboutR78-h82; GSK3 sites at about T23-S27, S99-v103, T113-S117, S132-t136,T136-S140, T199-p203, T201-p205, T297-S301, and S301-1305; SigPase sitesat about A195-A197 and A278-A280; Tyr kinase site at about D70-Y74; cAMPPK sites at about R78-T81, K96-S99, K157-S160, and K312-S315; Ca++phosphatase sites at about T149-E151 and T309-E311; cyt C Me site atabout V235-F238; myristoly sites at about F3-G7 and G329-T333; Nglycosylation sites at about N25-S27, N33-T35, N79-T81, and N147-T149;phos2 sites at about K28/S30, K96/S98, K96/S99, R104/S106, K206/S208,and K312/S315; PKC phosphorylation sites at about S76-R78, S99-K101, andS301-R303; SPKK sites at about S99-h103 and S301-m303; and Tyr Kinasesites at about R39-Y46.

In the IL-B50 sequence, the region from K97-K103 is known to be subjectto proteolysis, and mutations may be targeted to that region to protectthe ligand from proteolytic degradation. Thus, pharmacokineticproperties of the ligand may be modified, especially for the indicationsdescribed herein.

Segments with boundaries adjacent these positions will be particularlyuseful, as will polynucleotides encoding such segments. Mutagenesis inthese regions will be used to determine structure-activity relationship,particularly with the receptor, as provided herein.

As used herein, the term “primate IL-7Rα” shall be used to describe aprotein comprising a protein or peptide segment having or sharing theamino acid sequence shown in FIGS. 1A-1E (SEQ ID NO:2), or a substantialfragment thereof; but distinct from rodent sequences. The invention alsoincludes protein variations of the IL-7Rα allele whose sequence isprovided, e.g., a mutein agonist or antagonist. Typically, such agonistsor antagonists will exhibit less than about 10% sequence differences,and thus will often have between 1- and 11-fold substitutions, e.g., 2-,3-, 5-, 7-fold, and others. It also encompasses allelic and othervariants, e.g., natural polymorphic variants, of the protein described.“Natural” as used herein means unmodified by artifice, found, e.g., innatural sources. Typically, it will bind, when in a functional receptorcomplex, to ligand with high affinity, e.g., at least about 100 nM,usually better than about 30 nM, preferably better than about 10 nM, andmore preferably at better than about 3 nM. The term shall also be usedherein to refer to related naturally occurring forms, e.g., alleles,polymorphic variants, and metabolic variants of the primate protein.Corresponding meanings apply to uses of terms related to the Rδ2sequences.

This invention also encompasses proteins or peptides having amino acidsequence homology with combinations of amino acid sequences presented inFIGS. 1 and 2. In particular, it will include fusion constructs orproteins comprising segments from both of the sequences provided.

A substantial polypeptide “fragment”, or “segment”, is a stretch ofamino acid residues of at least about 8 amino acids, generally at least10 amino acids, more generally at least 12 amino acids, often at least14 amino acids, more often at least 16 amino acids, typically at least18 amino acids, more typically at least 20 amino acids, usually at least22 amino acids, more usually at least 24 amino acids, preferably atleast 26 amino acids, more preferably at least 28 amino acids, and, inparticularly preferred embodiments, at least about 30 or more aminoacids. Sequences of segments of different proteins can be compared toone another over appropriate length stretches.

Amino acid sequence homology, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. See, e.g., Needleham, et al., (1970) J. Mol. Biol. 48:443-453;Sankoff, et al., (1983) chapter one in Time Warps, String Edits, andMacromolecules: The Theory and Practice of Sequence Comparison,Addison-Wesley, Reading, Mass.; and software packages fromIntelliGenetics, Mountain View, Calif.; and the University of WisconsinGenetics Computer Group (GCG), Madison, Wis.; each of which isincorporated herein by reference. This changes when consideringconservative substitutions as matches. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. Homologous amino acid sequences are intended toinclude natural allelic and interspecies variations in the cytokinesequence. Typical homologous proteins or peptides will have from 50-100%homology (if gaps can be introduced), to 60-100% homology (ifconservative substitutions are included) with an amino acid sequencesegment of FIG. 1 or 2. Homology measures will be at least about 70%,generally at least 76%, more generally at least 81%, often at least 85%,more often at least 88%, typically at least 90%, more typically at least92%, usually at least 94%, more usually at least 95%, preferably atleast 96%, and more preferably at least 97%, and in particularlypreferred embodiments, at least 98% or more. The degree of homology willvary with the length of the compared segments. Homologous proteins orpeptides, such as the allelic variants, will share most biologicalactivities with the embodiments shown in FIGS. 1 and 2. As used herein,the term “biological activity” is used to describe, without limitation,effects on immune or other cell proliferation, differentiation,induction of cytokines or chemokines (e.g., TARC, PARC, MDC, MIP3-b,etc.), effects on STAT3 and/or STAT5 mediated signal transduction,antigen presentation effects, changes in cell surface moleculeexpression, and Th2 specific activities.

The terms ligand, agonist, antagonist, and analog of these receptors,particularly the functional complex, include molecules that modulate thecharacteristic cellular responses to the IL-B50, as well as moleculespossessing the more standard structural binding competition features ofligand-receptor interactions, e.g., where the receptor is a naturalreceptor or an antibody. The cellular responses likely are mediatedthrough binding of IL-B50 to cellular receptors, as described. Also, aligand is a molecule which serves either as a natural ligand to whichsaid receptor, or an analog thereof, binds, or a molecule which is afunctional analog of the natural ligand. The functional analog may be aligand with structural modifications, or may be a wholly unrelatedmolecule which has a molecular shape which interacts with theappropriate ligand binding determinants. The ligands may serve asagonists or antagonists, see, e.g., Goodman, et al. (eds.) (1990)Goodman & Gilman's: The Pharmacological Bases of Therapeutics, PergamonPress, New York.

Rational drug design may also be based upon structural studies of themolecular shapes of a receptor or antibody and other effectors orligands. Effectors may be other proteins which mediate other functionsin response to ligand binding, or other proteins which normally interactwith the receptor. One means for determining which sites interact withspecific other proteins is a physical structure determination, e.g.,x-ray crystallography or 2 dimensional NMR techniques. These willprovide guidance as to which amino acid residues form molecular contactregions. For a detailed description of protein structural determination,see, e.g., Blundell and Johnson (1976) Protein Crystallography, AcademicPress, New York, which is hereby incorporated herein by reference.

II. Activities

The IL-B50 proteins have a number of different biological activitiesbased on coexpression of IL-7Rα and Rδ2, e.g., in the immune system, andinclude proliferative, developmental, or physiological functions, inparticular of lymphoid lineage cells, e.g., macrophages or dendriticcells. The IL-B50 proteins are homologous to other IL-7 ligand familyproteins, but each have structural differences. For example, humanIL-B50 shows 43% amino acid sequence identity to mouse TSLP.Additionally, the human receptor subunit Rδ2 displays 39% amino acidsequence identity to mouse TSLPR.

The mouse IL-B50 molecule has the ability to stimulate TARC productionand various Th2 specific cytokines. The signaling pathway seems to useSTAT3 and/or STAT5, and sends proliferation or differentiation signals.Differentiation tends to result in limitation of proliferation, and viceversa. Differentiation typically results in changes in cell surfacemarker expression.

As shown herein, human IL-B50 improves dendritic cell survival incultures, upregulates the expression of costimulatory molecules andadhesion molecules, including HLA-DR, CD40, CD80, CD86, CD11a, CD18 andCD83, induces dendritic cells to produce the chemokines TARC, PARC andMDC, and strongly promotes the capacity of dendritic cells to inducenaïve T cells to proliferate and to produce cytokines IL-4, IL-13, andTNF-alpha. Additionally, IL-B50 has a synergistic effect withCD40-ligand and LPS in activated dendritic cells to upregulatecostimulatory molecules CD40, CD80 and CD86. IL-B50 also stronglyenhances CD40-ligand-induced production of IL-12 by dendritic cells.

TARC, PARC and MDC are all notably ligands for CCR4, a chemokinereceptor predominantly found on Th2-type lymphocytes. Thus, IL-B50 canactivate myeloid cells, such as monocytes, to release chemokines thatmay attract effector cells with a Th2 phenotype. As shown in theexamples below, IL-B50-induced expression of TARC was very strong in theCD11c⁺ subset of DCs. This subset, representing less than 1% ofmononuclear cells in the blood, normally differentiates into mature DCsin response to inflammatory stimuli. The expression of TARC in thesecells was accompanied by a dramatic enhancement of their maturation asevidenced by the strong induction of the costimulatory molecules CD40and CD80. These results indicate that this DC subset stimulated withIL-B50 could be a potent inducer of primary T-cell-mediated immuneresponses. Indeed, CD11+ DCs cultured in the presence of IL-B50 are muchmore potent in their capacity to elicit the proliferation of naïve Tcells as compared to DC cultured in medium.

Dendritic cells are professional antigen presenting cells, which arecapable of inducing primary antigen-specific T cell-mediated immuneresponses. Dendritic cells play a critical role in initiating immuneresponses against tumors and infectious microorganisms. Dendritic cellsare also involved in autoimmune diseases, allergic diseases,graft-versus-host disease and rejection of solid organ transplants.Therefore, enhancing dendritic cell function allows for treatment oftumors and infectious diseases. Similarly, blocking dendritic cellfunction provides therapies for autoimmune diseases, allergic diseases,graft-versus-host diseases and transplantation associated rejection.

Thus, IL-B50 may be used in enhancing dendritic cell function intreating cancers and infectious diseases and IL-B50 antagonists may beused in blocking the function of dendritic cells in treating autoimmunediseases, allergic diseases, graft-versus-host diseases andtransplantation associated rejection. The elucidation of the IL-B50receptor subunits, therefore, allows for the identification of agonistsand antagonists of ILB-50 for use in treating the aforementioneddiseases.

The present disclosure also describes new assays for activitiesdescribed for these molecules. Corresponding activities should be foundin other mammalian systems, including primates. The new IL-7-likemolecules produced by recombinant means exhibit a biological activity ofmodulating lymphoid lineage cells. Furthermore, there is substantiallikelihood of synergy with other IL-7 related agonists or antagonists.It is likely that the receptors, which are expected to include multipledifferent polypeptide chains, exhibit species specificity for theircorresponding ligands.

III. Nucleic Acids

This invention contemplates use of isolated nucleic acid or fragments,e.g., which encode these or closely related proteins, or fragmentsthereof, e.g., fusion proteins or coordinately expressed or combinationexpression constructs.

The term “isolated nucleic acid or fragments” as used herein means anucleic acid, e.g., a DNA or RNA molecule, that is not immediatelycontiguous with sequences present in the naturally occurring genome ofthe organism from which it is derived. Thus, the term describes, e.g., anucleic acid that is incorporated into a vector, such as a plasmid orviral vector; a nucleic acid that is incorporated into the genome of aheterologous cell (or the genome of homologous cell, but at a sitedifferent from that at which it normally occurs); and a nucleic acidthat exists as a separate molecule, e.g., a DNA fragment produced by PCRamplification or restriction enzyme digestion, or an RNA moleculeproduced by in vitro transcription. The term also describes arecombinant (e.g., genetically engineered) nucleic acid that forms partof a hybrid gene encoding additional polypeptide sequences that can beused, e.g., in the production of a fusion protein. In addition, thisinvention embodies any engineered or nucleic acid molecule created byartifice that encodes a biologically active protein or polypeptidehaving characteristic IL-B50 receptor activity. Typically, the nucleicacid is capable of hybridizing, under appropriate conditions, withnucleic acid sequence segments shown in FIGS. 1 and 2. Further, thisinvention covers the use of isolated or recombinant nucleic acid, orfragments thereof, which encode proteins having fragments which arehomologous to the newly disclosed receptor complex proteins. Theisolated nucleic acids can have the respective regulatory sequences inthe 5′ and 3′ flanks, e.g., promoters, enhancers, poly-A additionsignals, and others from the natural gene.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially pure, e.g., separated from othercomponents which naturally accompany a native sequence, such asribosomes, polymerases, and flanking genomic sequences from theoriginating species. The term embraces a nucleic acid sequence which hasbeen removed from its naturally occurring environment, and includesrecombinant or cloned DNA isolates, which are thereby distinguishablefrom naturally occurring compositions, and chemically synthesizedanalogs or analogs biologically synthesized by heterologous systems. Asubstantially pure molecule includes isolated forms of the molecule,either completely or substantially pure.

An isolated nucleic acid will generally be a homogeneous composition ofmolecules, but will, in some embodiments, contain heterogeneity,preferably minor. This heterogeneity is typically found at the polymerends or portions not critical to a desired biological function oractivity.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence. Typically this intervention involves in vitromanipulation, although under certain circumstances it may involve moreclassical animal breeding techniques. Alternatively, it can be a nucleicacid made by generating a sequence comprising fusion of two fragmentswhich are not naturally contiguous to each other, but is meant toexclude products of nature, e.g., naturally occurring mutants as foundin their natural state. Thus, e.g., products made by transforming cellswith any unnaturally occurring vector is encompassed, as are nucleicacids comprising sequence derived using any synthetic oligonucleotideprocess. Such a process is often done to replace a codon with aredundant codon encoding the same or a conservative amino acid, whiletypically introducing or removing a restriction enzyme sequencerecognition site. Alternatively, the process is performed to jointogether nucleic acid segments of desired functions to generate a singlegenetic entity comprising a desired combination of functions not foundin the commonly available natural forms, e.g., encoding a fusionprotein. Restriction enzyme recognition sites are often the target ofsuch artificial manipulations, but other site specific targets, e.g.,promoters, DNA replication sites, regulation sequences, controlsequences, or other useful features may be incorporated by design. Asimilar concept is intended for a recombinant, e.g., fusion,polypeptide. This will include a dimeric repeat. Specifically includedare synthetic nucleic acids which, by genetic code redundancy, encodesimilar combination polypeptides to fragments of the receptor subunitsand fusions of sequences from various different receptors or relatedmolecules, e.g., growth factors.

A “fragment” in a nucleic acid context is a contiguous segment of atleast about 17 nucleotides, generally at least 21 nucleotides, moregenerally at least 25 nucleotides, ordinarily at least 30 nucleotides,more ordinarily at least 35 nucleotides, often at least 39 nucleotides,more often at least 45 nucleotides, typically at least 50 nucleotides,more typically at least 55 nucleotides, usually at least 60 nucleotides,more usually at least 66 nucleotides, preferably at least 72nucleotides, more preferably at least 79 nucleotides, and inparticularly preferred embodiments will be at least 85 or morenucleotides including, e.g., 100, 150, 200, 250, etc. Typically,fragments of different genetic sequences can be compared to one anotherover appropriate length stretches, particularly defined segments such asthe domains described below.

A nucleic acid which codes for an IL-B50 receptor complex will beparticularly useful to identify genes, mRNA, and cDNA species which codefor itself or closely related proteins, as well as DNAs which code forpolymorphic, allelic, or other genetic variants, e.g., from differentindividuals or related species. Preferred probes for such screens arethose regions of the interleukin which are conserved between differentpolymorphic variants or which contain nucleotides which lackspecificity, and will preferably be full length or nearly so. In othersituations, polymorphic variant specific sequences will be more useful.

This invention further covers recombinant nucleic acid molecules andfragments having a nucleic acid sequence identical to or highlyhomologous to the isolated DNA set forth herein. In particular, thesequences will often be operably linked to DNA segments which controltranscription, translation, and DNA replication. These additionalsegments typically assist in expression of the desired nucleic acidsegment.

Homologous nucleic acid sequences, when compared to one another or tothe sequences shown in FIG. 1 or 2, exhibit significant similarity. Thestandards for homology in nucleic acids are either measures for homologygenerally used in the art by sequence comparison or based uponhybridization conditions. Comparative hybridization conditions aredescribed in greater detail below.

Substantial identity in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 60% of thenucleotides, generally at least 66%, ordinarily at least 71%, often atleast 76%, more often at least 80%, usually at least 84%, more usuallyat least 88%, typically at least 91%, more typically at least about 93%,preferably at least about 95%, more preferably at least about 96 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides, including, e.g., segments encoding structural domainssuch as the segments described below. Alternatively, substantialidentity will exist when the segments will hybridize under selectivehybridization conditions, to a strand or its complement, typically usinga sequence derived from the sequences depicted in FIGS. 1 and 2.Typically, selective hybridization will occur when there is at leastabout 55% homology over a stretch of at least about 14 nucleotides, moretypically at least about 65%, preferably at least about 75%, and morepreferably at least about 90%. See, Kanehisa (1984) Nuc. Acids Res.12:203-213. The length of homology comparison, as described, may be overlonger stretches, and in certain embodiments will be over a stretch ofat least about 17 nucleotides, generally at least about 20 nucleotides,ordinarily at least about 24 nucleotides, usually at least about 28nucleotides, typically at least about 32 nucleotides, more typically atleast about 40 nucleotides, preferably at least about 50 nucleotides,and more preferably at least about 75 to 100 or more nucleotides.

Stringent conditions, in referring to homology in the hybridizationcontext, will be stringent combined conditions of salt, temperature,organic solvents, and other parameters typically controlled inhybridization reactions. Stringent temperature conditions will usuallyinclude temperatures in excess of about 30 degrees C., more usually inexcess of about 37 degrees C., typically in excess of about 45 degreesC., more typically in excess of about 55 degrees C., preferably inexcess of about 65 degrees C., and more preferably in excess of about 70degrees C. Stringent salt conditions will ordinarily be less than about500 mM, usually less than about 400 mM, more usually less than about 300mM, typically less than about 200 mM, preferably less than about 100 mM,and more preferably less than about 80 mM, even down to less than about20 mM. Certain detergents or destabilizing reagents may be added, e.g.,formamide at 50%, etc. However, the combination of parameters is muchmore important than the measure of any single parameter. See, e.g.,Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370, which is herebyincorporated herein by reference.

The isolated DNA can be readily modified by nucleotide substitutions,nucleotide deletions, nucleotide insertions, and inversions ofnucleotide stretches. These modifications result in novel DNA sequenceswhich encode this protein or its derivatives. These modified sequencescan be used to produce mutant proteins (muteins) or to enhance theexpression of variant species. Enhanced expression may involve geneamplification, increased transcription, increased translation, and othermechanisms. Such mutant receptor-like derivatives include predeterminedor site-specific mutations of the protein or its fragments, includingsilent mutations using genetic code degeneracy. “Mutant IL-B50 receptor”as used herein encompasses a polypeptide otherwise falling within thehomology definition of the receptor as set forth above, but having anamino acid sequence which differs from that of other IL-7 receptor-likeproteins as found in nature, whether by way of deletion, substitution,or insertion. In particular, “site specific mutant IL-B50 receptor”encompasses a protein having substantial homology with a protein shownin FIGS. 1 and 2, and typically shares most of the biological activitiesof the form disclosed herein.

Although site specific mutation sites are predetermined, mutants neednot be site specific. Mammalian IL-B50 receptor mutagenesis can beachieved by making amino acid insertions or deletions in the gene,coupled with expression. Substitutions, deletions, insertions, or anycombinations may be generated to arrive at a final construct. Insertionsinclude amino- or carboxy-terminal fusions. Random mutagenesis can beconducted at a target codon and the expressed mammalian IL-B50 receptormutants can then be screened for the desired activity. Methods formaking substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis. See also Sambrook, et al. (1989) and Ausubel, et al. (1987and periodic Supplements).

The mutations in the DNA normally should not place coding sequences outof reading frames and preferably will not create complementary regionsthat could hybridize to produce secondary mRNA structure such as loopsor hairpins.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Polymerase chain reaction (PCR) techniques can often be applied inmutagenesis. Alternatively, mutagenesis primers are commonly usedmethods for generating defined mutations at predetermined sites. See,e.g., Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (eds. 1995) PCR Primer: A Laboratory Manual Cold Spring HarborPress, CSH, NY.

IV. Proteins, Peptides

As described above, the present invention encompasses primate IL-B50receptor, e.g., whose sequences are disclosed in FIGS. 1 and 2, anddescribed above. Allelic and other variants are also contemplated,including, e.g., fusion proteins combining portions of such sequenceswith others, including epitope tags and functional domains.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these primate proteins.A heterologous fusion protein is a fusion of proteins or segments whichare naturally not normally fused in the same manner. Thus, the fusionproduct of a growth factor receptor with a cytokine receptor is acontinuous protein molecule having sequences fused in a typical peptidelinkage, typically made as a single translation product and exhibitingproperties, e.g., antigenicity, derived from each source peptide. Asimilar concept applies to heterologous nucleic acid sequences.

In addition, new constructs may be made from combining similarfunctional or structural domains from other related proteins, e.g.,growth factors or other cytokines. For example, receptor-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992,each of which is incorporated herein by reference. Thus, new chimericpolypeptides exhibiting new combinations of specificities will resultfrom the functional linkage of receptor-binding specificities. Forexample, the ligand binding domains from other related receptormolecules may be added or substituted for other domains of these orrelated proteins. The resulting protein will often have hybrid functionand properties. For example, a fusion protein may include a labelingepitope which may serve to provide diagnostic locatability of the fusionprotein for histology or other methods.

Candidate fusion partners and sequences can be selected from varioussequence data bases, e.g., GenBank, c/o IntelliGenetics, Mountain View,Calif.; and BCG, University of Wisconsin Biotechnology Computing Group,Madison, Wis., which are each incorporated herein by reference.

“Derivatives” of the mammalian IL-B50 receptor include amino acidsequence mutants, glycosylation variants, metabolic derivatives andcovalent or aggregative conjugates with other chemical moieties.Covalent derivatives can be prepared by linkage of functionalities togroups which are found in the IL-B50 receptor amino acid side chains orat the N- or C-termini, e.g., by means which are well known in the art.These derivatives can include, without limitation, aliphatic esters oramides of the carboxyl terminus, or of residues containing carboxyl sidechains, O-acyl derivatives of hydroxyl group-containing residues, andN-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues, e.g., lysine or arginine. Acyl groups are selectedfrom the group of alkyl-moieties including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

A major group of derivatives are covalent conjugates of the interleukinor fragments thereof with other proteins of polypeptides. Thesederivatives can be synthesized in recombinant culture such as N- orC-terminal fusions or by the use of agents known in the art for theirusefulness in cross-linking proteins through reactive side groups.Preferred derivatization sites with cross-linking agents are at freeamino groups, carbohydrate moieties, and cysteine residues.

Fusion polypeptides between the receptors and other homologous orheterologous proteins are also provided. Homologous polypeptides may befusions between different receptors, resulting in, for instance, ahybrid protein exhibiting binding specificity for multiple differentligands, or a receptor which may have broadened or weakened specificityof binding to its ligand. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of areceptor, e.g., a ligand-binding segment, so that the presence orlocation of a desired receptor may be easily determined. See, e.g.,Dull, et al., U.S. Pat. No. 4,859,609, which is hereby incorporatedherein by reference. Other gene fusion partners includeglutathione-S-transferase (GST), bacterial β-galactosidase, trpE,Protein A, β-lactamase, alpha amylase, alcohol dehydrogenase, yeastalpha mating factor, or other cytokine receptors. See, e.g., Godowski,et al. (1988) Science 241:812-816.

The phosphoramidite method described by Beaucage and Carruthers (1981)Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNAfragments. A double stranded fragment will often be obtained either bysynthesizing the complementary strand and annealing the strand togetherunder appropriate conditions or by adding the complementary strand usingDNA polymerase with an appropriate primer sequence.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation,PEGylation, or the addition or removal of other moieties, particularlythose which have molecular shapes similar to phosphate groups. In someembodiments, the modifications will be useful labeling reagents, orserve as purification targets, e.g., affinity ligands.

Fusion proteins will typically be made by either recombinant nucleicacid methods or by synthetic polypeptide methods. Techniques for nucleicacid manipulation and expression are described generally, e.g., inSambrook, et al. (1989) Molecular Cloning: A LaboratorEy Manual (2ded.), Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al.(eds. 1987 and periodic supplements) Current Protocols in MolecularBiology, Greene/Wiley, New York, which are each incorporated herein byreference. Techniques for synthesis of polypeptides are described, e.g.,in Merrifield (1963) J. Amer. Chem. Soc. 85:2149-2156; Merrifield (1986)Science 232: 341-347; and Atherton, et al. (1989) Solid Phase PeptideSynthesis: A Practical Approach, IRL Press, Oxford; each of which isincorporated herein by reference. See also Dawson, et al. (1994) Science266:776-779 for methods to make larger polypeptides.

The term “substantially purified” as used herein refers to a molecule,such as a peptide that is substantially free of other proteins, lipids,carbohydrates, nucleic acids, or other biological materials with whichit is naturally associated. For example, a substantially pure molecule,such as a polypeptide, can be at least 60%, by dry weight, the moleculeof interest. One skilled in the art can purify complexes comprising thedescribed sequences using standard protein purification methods and thepurity of the polypeptides can be determined using standard methodsincluding, e.g., polyacrylamide gel electrophoresis (e.g., SDS-PAGE),column chromatography (e.g., high performance liquid chromatography(HPLC)), and amino-terminal amino acid sequence analysis.

Changes in the amino acid sequence of the receptor components of thecomplex are contemplated in the present invention. The IL-7Rα or Rδ2 canbe altered by changing the nucleic acid sequence encoding the proteins.Preferably, only conservative amino acid alterations are undertaken,using amino acids that have the same or similar properties. Illustrativeamino acid substitutions include the changes of: alanine to serine;arginine to lysine; asparagine to glutamine or histidine; aspartate toglutamate; cysteine to serine; glutamine to asparagine; glutamate toaspartate; glycine to proline; histidine to asparagine or glutamine;isoleucine to leucine or valine; leucine to valine or isoleucine; lysineto arginine, glutamine, or glutamate; methionine to leucine orisoleucine; phenylalanine to tyrosine, leucine or methionine; serine tothreonine; threonine to serine; tryptophan to tyrosine; tyrosine totryptophan or phenylalanine; valine to isoleucine or leucine.

Additionally, other variants and fragments of receptor complexes can beused in the present invention. Variants include analogs, homologues,derivatives, muteins, and mimetics of the receptor complexes orfragments that retain or enhance the ability to block binding betweenIL-B50 and a target receptor. The variants and fragments can begenerated directly from sequences provided, by chemical modification, byproteolytic enzyme digestion, or by combinations thereof. Additionally,genetic engineering techniques, as well as methods of synthesizingpolypeptides directly from amino acid residues, can be employed.

Non-peptide compounds that mimic the binding and function of IL-B50(“mimetics”) can be produced by the approach outlined in Saragovi, etal. (1991) Science 253:792-95. Mimetics are molecules which mimicelements of protein secondary structure. See, e.g., Johnson et al.,“Peptide Turn Mimetics,” in Pezzuto, et al. (eds. 1993) Biotechnologyand Pharmacy, Chapman and Hall, New York. The underlying rationalebehind the use of peptide mimetics is that the peptide backbone ofproteins exists chiefly to orient amino acid side chains in such a wayas to facilitate molecular interactions. For the purposes of the presentinvention, appropriate mimetics can be considered to be the equivalentof IL-B50 itself.

Variants and fragments also can be created by recombinant techniquesemploying genomic or cDNA cloning methods. Site-specific andregion-directed mutagenesis techniques can be employed. See, e.g., vol.1, ch. 8 in Ausubel, et al. (eds. 1989 and periodic updates) CurrentProtocols in Molecular Biology Wiley and Sons; and Oxender and Fox(eds.) Protein Engineering Liss, Inc. In addition, linker-scanning andPCR-mediated techniques can be employed for mutagenesis. See, e.g.,Erlich (ed. 1989) PCR Technology Stockton Press. Protein sequencing,structure and modeling approaches for use with any of the abovetechniques are disclosed, e.g., in Oxender and Fox (eds.) ProteinEngineering Liss, Inc; and Ausubel, et al. (eds. 1989 and periodicupdates) Current Protocols in Molecular Biology Wiley and Sons.

This invention also contemplates the use of derivatives of IL-B50receptor other than variations in amino acid sequence or glycosylation.Such derivatives may involve covalent or aggregative association withchemical moieties. These derivatives generally fall into three classes:(1) salts, (2) side chain and terminal residue covalent modifications,and (3) adsorption complexes, e.g., with cell membranes. Such covalentor aggregative derivatives are useful as immunogens, as reagents inimmunoassays, in enhancing pharmacokinetic properties of the protein, orin purification methods such as for affinity purification of a receptoror other binding molecule, e.g., an antibody. For example, an IL-B50receptor can be immobilized by covalent bonding to a solid support suchas cyanogen bromide-activated SEPHAROSE, by methods which are well knownin the art, or adsorbed onto polyolefin surfaces, with or withoutglutaraldehyde cross-linking, for use in the assay or purification ofIL-B50 receptor, antibodies, or other similar molecules. The IL-B50 canalso be labeled with a detectable group, e.g., radio-iodinated by thechloramine T procedure, covalently bound to rare earth chelates, orconjugated to another fluorescent moiety for use in diagnostic assays.

A receptor complex of this invention can be used as an immunogen for theproduction of antisera or antibodies specific, e.g., for the complexcomprising both protein components. The purified interleukin can be usedto screen monoclonal antibodies or antigen-binding fragments prepared byimmunization with various forms of impure preparations containing theprotein. In particular, the term “antibodies” also encompasses antigenbinding fragments of natural antibodies. The purified interleukin canalso be used as a reagent to detect any antibodies generated in responseto the presence of elevated levels of expression, or immunologicaldisorders which lead to antibody production to the endogenous cytokine.Additionally, receptor complex fragments may also serve as immunogens toproduce the antibodies of the present invention, as describedimmediately below. For example, this invention contemplates antibodieshaving binding affinity to or being raised against a complex comprisingthe amino acid sequences shown in FIGS. 1 and 2, fragments thereof, orhomologous peptides. In particular, this invention contemplatesantibodies having binding affinity to, or having been raised against,specific fragments which are predicted to be, or actually are, exposedat the exterior protein surface of the native receptor.

The blocking of physiological response to the ligand may result from theinhibition of binding of the ligand to the receptor, likely throughcompetitive inhibition. Thus, in vitro assays of the present inventionwill often use antibodies or receptor binding segments of theseantibodies, or fragments attached to solid phase substrates. Theseassays will also allow for the diagnostic determination of the effectsof either binding region mutations and modifications, or receptormutations and modifications.

This invention also contemplates the use of competitive drug screeningassays, e.g., where neutralizing antibodies to the receptor complex orfragments compete with a test compound for binding. In this manner, theneutralizing antibodies or fragments can be used to detect the presenceof a polypeptide which shares one or more binding sites to a receptorand can also be used to occupy binding sites on a receptor that mightotherwise bind a cytokine, e.g., IL-B50.

V. Making Nucleic Acids and Protein

DNA which encodes the proteins or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.Natural sequences can be isolated using standard methods and thesequences provided herein, e.g., in FIGS. 1 and 2. Other speciescounterparts, e.g., primate, can be identified by hybridizationtechniques, or by various PCR techniques, combined with or by searchingin sequence databases.

These DNAs can be expressed in a wide variety of host cells for thesynthesis and formation of the functional receptor complex, orfragments, which can in turn, e.g., be used to generate polyclonal ormonoclonal antibodies; for binding studies; for construction andexpression of modified agonist/antagonist molecules; forstructure/function studies; and for screening studies. A variant or itsfragments can be expressed in host cells that are transformed ortransfected with appropriate expression vectors. These molecules can besubstantially free of protein or cellular contaminants, other than thosederived from the recombinant host, and therefore are particularly usefulin pharmaceutical compositions when combined with a pharmaceuticallyacceptable carrier and/or diluent. The proteins, or portions thereof,may be expressed as fusions with other proteins or using combiningmotifs, e.g., leucine zippers, to form a soluble receptor complex.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired receptor gene or its fragments, usually operablylinked to suitable genetic control elements that are recognized in asuitable host cell. These control elements are capable of effectingexpression within a suitable host. The specific type of control elementsnecessary to effect expression will depend upon the eventual host cellused. Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently of thehost cell.

The vectors of this invention include those which contain DNA whichencodes these proteins, as described, or a fragment comprising sequencefrom both subunits, typically encoding a functionally, e.g.,biologically active, equivalent polypeptide. The DNA can be under thecontrol of a viral promoter and can encode a selection marker. Thisinvention further contemplates use of such expression vectors which arecapable of expressing eukaryotic cDNA coding for such a protein orcomplex in a prokaryotic or eukaryotic host, where the vector iscompatible with the host and where the eukaryotic cDNA coding for thereceptor is inserted into the vector such that growth of the hostcontaining the vector expresses the cDNA in question. Usually,expression vectors are designed for stable replication in their hostcells or for amplification to greatly increase the total number ofcopies of the desirable gene per cell. It is not always necessary torequire that an expression vector replicate in a host cell, e.g., it ispossible to effect transient expression of the receptor or its fragmentsin various hosts using vectors that do not contain a replication originthat is recognized by the host cell. It is also possible to use vectorsthat cause integration of the protein encoding portion or its fragmentsinto the host DNA by recombination, e.g., downstream of heterologouspromoters, or integration of heterologous promoters upstream fromendogenous genes.

Vectors, as used herein, comprise plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles, e.g., which may enablethe integration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector but other forms of vectors which serve anequivalent function and which are, or become, known in the art aresuitable for use herein. See, e.g., Pouwels, et al. (1985 andSupplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., andRodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, which are incorporatedherein by reference.

Transformed cells are cells, preferably mammalian, that have beentransformed, transfected, or infected with vectors, e.g., constructedusing recombinant DNA techniques. Transformed host cells usually expressthe desired protein or its fragments, but for purposes of cloning,amplifying, and manipulating its DNA, do not need to express the subjectprotein. This invention further contemplates culturing transformed cellsin a nutrient medium, thus permitting the cells to express the protein,including production of soluble receptor complexes or ligand.

For purposes of this invention, nucleic acid sequences are operablylinked when they are functionally related to each other. For example,DNA for a pre-sequence or secretory leader is operably linked to apolypeptide if it is expressed as a pre-protein or participates indirecting the polypeptide to the cell membrane or in secretion of thepolypeptide. A promoter is operably linked to a coding sequence if itpromotes the transcription of the polypeptide; a ribosome binding siteis operably linked to a coding sequence if it is positioned to inducetranslation. Usually, operably linked means contiguous and in readingframe, however, certain genetic elements such as repressor genes are notcontiguously linked but still bind to operator sequences that in turncontrol expression.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia species, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A common vector for amplifying DNA is pBR322 or many of itsderivatives. Vectors that can be used to express the receptor or itsfragments include, but are not limited to, such vectors as thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius, et al. (1988) “ExpressionVectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, inRodriguez and Denhardt (eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses, Buttersworth, Boston, Chapter 10, pp. 205-236,which is incorporated herein by reference.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith IL-B50 receptor sequence containing vectors. For purposes of thisinvention, the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used to generically represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the receptor or its fragments, and sequences for translationtermination, polyadenylation, and transcription termination. Suitableexpression vectors for yeast include such constitutive promoters as3-phosphoglycerate kinase and various other glycolytic enzyme genepromoters or such inducible promoters as the alcohol dehydrogenase 2promoter or metallothionine promoter. Suitable vectors includederivatives of the following types: self-replicating low copy number(such as the YRp-series), self-replicating high copy number (such as theYEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are normally the preferred hostcells for expression of the functionally active interleukin protein. Inprinciple, any higher eukaryotic tissue culture cell line is workable,e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred. Transformation or transfection and propagation of such cellshas become a routine procedure. Examples of useful cell lines includeHeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney(BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS)cell lines. Expression vectors for such cell lines usually include anorigin of replication, a promoter, a translation initiation site, RNAsplice sites (if genomic DNA is used), a polyadenylation site, and atranscription termination site. These vectors also usually contain aselection gene or amplification gene. Suitable expression vectors may beplasmids, viruses, or retroviruses carrying promoters derived, e.g.,from such sources as from adenovirus, SV40, parvoviruses, vacciniavirus, or cytomegalovirus. Representative examples of suitableexpression vectors include pCDNA1; pCD, see Okayama, et al. (1985) Mol.Cell Biol. 5:1136-1142; pMC1neo PolyA, see Thomas, et al. (1987) Cell51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.

For secreted proteins, an open reading frame usually encodes apolypeptide that consists of a mature or secreted product covalentlylinked at its N-terminus to a signal peptide. The signal peptide istypically cleaved prior to secretion of the mature, or active,polypeptide. The cleavage site can be predicted with a high degree ofaccuracy from empirical rules, e.g., von-Heijne (1986) Nucleic AcidsResearch 14:4683-4690, and the precise amino acid composition of thesignal peptide does not appear to be critical to its function. See,e.g., Randall, et al. (1989) Science 243:1156-1159; and Kaiser et al.(1987) Science 235:312-317.

It will often be desired to express these polypeptides in a system whichprovides a specific or defined glycosylation pattern. In this case, theusual pattern will be that provided naturally by the expression system.However, the pattern will be modifiable by exposing the polypeptide,e.g., an unglycosylated form, to appropriate glycosylating proteinsintroduced into a heterologous expression system. For example, thereceptor genes may be co-transformed with one or more genes encodingmammalian or other glycosylating enzymes. Using this approach, certainmammalian glycosylation patterns will be achievable in prokaryote orother cells.

The source of receptor complex can be a eukaryotic or prokaryotic hostexpressing recombinant receptor subunit DNA, such as is described above.The source can also be a cell line such as mouse Swiss 3T3 fibroblasts,but other mammalian cell lines are also contemplated by this invention,with the preferred cell line being from the human species.

Now that the entire sequence and components of the functional receptorcomplex is known, the human receptor complex, fragments, or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis, Springer-Verlag, New York; and Bodanszky (1984) ThePrinciples of Peptide Synthesis, Springer-Verlag, New York; all of eachwhich are incorporated herein by reference. For example, an azideprocess, an acid chloride process, an acid anhydride process, a mixedanhydride process, an active ester process (e.g., p-nitrophenyl ester,N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazoleprocess, an oxidative-reductive process, or a dicyclohexylcarbodiimide(DCCD)/additive process can be used. Solid phase and solution phasesyntheses are both applicable to the foregoing processes.

The receptor subunit proteins, fragments, or derivatives are suitablyprepared in accordance with the above processes as typically employed inpeptide synthesis, generally either by a so-called stepwise processwhich comprises condensing an amino acid to the terminal amino acid, oneby one in sequence, or by coupling peptide fragments to the terminalamino acid. Amino groups that are not being used in the couplingreaction typically must be protected to prevent coupling at an incorrectlocation.

If a solid phase synthesis is adopted, the C-terminal amino acid isbound to an insoluble carrier or support through its carboxyl group. Theinsoluble carrier is not particularly limited as long as it has abinding capability to a reactive carboxyl group. Examples of suchinsoluble carriers include halomethyl resins, such as chloromethyl resinor bromomethyl resin, hydroxymethyl resins, phenol resins,tert-alkyloxycarbonylhydrazidated resins, and the like.

An amino group-protected amino acid is bound in sequence throughcondensation of its activated carboxyl group and the reactive aminogroup of the previously formed peptide or chain, to synthesize thepeptide step by step. After synthesizing the complete sequence, thepeptide is split off from the insoluble carrier to produce the peptide.This solid-phase approach is generally described by Merrifield, et al.(1963) in J. Am. Chem. Soc. 85:2149-2156, which is incorporated hereinby reference.

The prepared proteins and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, e.g., byextraction, precipitation, electrophoresis, various forms ofchromatography, and the like. The receptors of this invention can beobtained in varying degrees of purity depending upon its desired use.Purification can be accomplished by use of the protein purificationtechniques disclosed herein, see below, or by the use of the antibodiesherein described in methods of immunoabsorbant affinity chromatography.This immunoabsorbant affinity chromatography is typically carried out byfirst linking the antibodies to a solid support and then contacting thelinked antibodies with solubilized lysates of appropriate cells, lysatesof other cells expressing the receptor complexes, or lysates orsupernatants of cells producing the proteins as a result of DNAtechniques, see below.

Generally, the purified protein will be at least about 40% pure,ordinarily at least about 50% pure, usually at least about 60% pure,typically at least about 70% pure, more typically at least about 80%pure, preferable at least about 90% pure and more preferably at leastabout 95% pure, and in particular embodiments, 97%-99% or more. Puritywill usually be on a weight basis, but can also be on a molar basis.Different assays will be applied as appropriate.

VI. Antibodies

The term “antibody” or “antibody molecule” as used in this inventionincludes intact molecules as well as fragments thereof, such as Fab,F(ab′)₂, and Fv which are capable of selectively binding the epitopicdeterminant. These antibody fragments retain some ability to selectivelybind with its antigen or receptor and are defined as follows: (1) Fab,the fragment which contains a monovalent antigen-binding fragment of anantibody molecule can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)₂, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)₂ is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (5) Single chain antibody (“SCA”), definedas a genetically engineered molecule containing the variable region ofthe light chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule.

Methods of making these fragments are known in the art. See, e.g.,Harlow and Lane (current edition) Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York. Therefore, the phrase “antibodymolecule” in its various forms as used herein contemplates both anintact antibody (immunoglobulin) molecule and an immunologically activeportion of an antibody (immunoglobulin) molecule. Recombinant methodsmay be applied to make these fragments.

The term “monoclonal antibody” refers to a population of one species ofantibody molecule of antigen-specificity. A monoclonal antibody containsonly one species of antibody combining site capable of immunoreactingwith a particular antigen and thus typically displays a single bindingaffinity for that antigen. A monoclonal antibody may therefore contain abispecific antibody molecule having two antibody combining sites, eachimmunospecific for a different antigen. In one embodiment, the firstantibody molecule is affixed to a solid support.

As used in this invention, the term “epitope” means an antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics.

The word “complex” as used herein refers to the product of a specificbinding agent-target reaction. An exemplary complex is the combinationof two subunits to form a physiological complex of protein subunits, oran immunoreaction product formed by an antibody-antigen reaction.

The term “antigen” refers to a polypeptide or protein that is able tospecifically bind to (immunoreact with) an antibody and form animmunoreaction product (immunocomplex). The site on the antigen withwhich the antibody binds is referred to as an antigenic determinant orepitope, and the binding should be detectable, e.g., 2×, 5× or moreabove background.

A method of the invention for detection of antibodies that bind to novelepitopes in a sample is performed in vitro, e.g., in immunoassays inwhich the antibodies can be identified in liquid phase or bound to asolid phase carrier. In various embodiments, the method is performedwith a capture antibody bound to a solid support, and/or the captureantibody is a monoclonal antibody molecule. In other instances, the useof tetramer technology may be useful. See, e.g., Kelleher andRowland-Jones (2000) Curr. Op. Immunol. 12:370-374; Katz (1999) Biomol.Eng. 16:57-65; and Ogg and McMichael (1998) Curr. Op. Immunol.10:393-396.

Examples of types of immunoassays which can be utilized to detect novelantibodies in a sample, include competitive and non-competitiveimmunoassays in either a direct or indirect format. Examples of suchimmunoassays are the radioimmunoassay (RIA) and the sandwich(immunometric) assay. Detection of the antibodies can be done utilizingimmunoassays which are run in either the forward, reverse, orsimultaneous modes, including competition immunoassays andimmunohistochemical assays on physiological samples. Preferably, themethod of the invention utilizes a forward immunoassay. Those of skillin the art will know, or can readily discern, other immunoassay formatswithout undue experimentation.

Solid phase-bound antibody molecules are bound by adsorption from anaqueous medium, although other modes of affixation, such as covalentcoupling or other well known means of affixation to the solid matrix canbe used. Preferably, the first antibody molecule is bound to a supportbefore forming an immunocomplex with antigen, however, the immunocomplexcan be formed prior to binding the complex to the solid support.

Non-specific protein binding sites on the surface of the solid phasesupport are preferably blocked. After adsorption of solid phase-boundantibodies, an aqueous solution of a protein free from interference withthe assay such as bovine, horse, or other serum albumin that is alsofree from contamination with the antigen is admixed with the solid phaseto adsorb the admixed protein onto the surface of theantibody-containing solid support at protein binding sites on thesurface that are not occupied by the antibody molecule.

A typical aqueous protein solution contains about 2-10 weight percentbovine serum albumin in PBS at a pH of about 7-8. The aqueous proteinsolution-solid support mixture is typically maintained for a time periodof at least one hour at a temperature of about 4-37 degrees C. and theresulting solid phase is thereafter rinsed free of unbound protein.

The first preselected antibody can be bound to many different carriersand used to detect novel epitope binding antibodies in a sample.Examples of well-known carriers include glass, polystyrene,polypropylene, polyethylene, dextran, nylon, amyloses, natural andmodified celluloses, polyacrylamides, agaroses, and magnetite. Thenature of the carrier can be either soluble or insoluble for purposes ofthe invention. Those skilled in the art will know of other suitablecarriers for binding antibodies, or will be able to ascertain such,using routine experimentation.

In addition, if desirable, an antibody for detection in theseimmunoassays can be detectably labeled in various ways. There are manydifferent labels and methods of labeling known. Examples of the types oflabels which can be used in the present invention include, e.g.,enzymes, radioisotopes, fluorescent compounds, colloidal metals,chemiluminescent compounds, and bio-luminescent compounds. Many othersuitable labels exist for binding to the monoclonal antibodies of theinvention

Antibodies which bind to IL-B50 receptor complex can be prepared usingan intact polypeptide or fragments containing peptides of interest asthe immunizing antigen. The polypeptide or a peptide used to immunize ananimal can be derived from translated cDNA or chemical synthesis whichcan be conjugated to a carrier protein, if desired. Such commonly usedcarriers which are chemically coupled to the peptide include keyholelimpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), andtetanus toxoid. The coupled peptide is then used to immunize the hostanimal, e.g., a mouse, a rat, or a rabbit.

If desired, polyclonal or monoclonal antibodies can be further purified,e.g., by binding to and elution from a matrix to which is bound theantigen to which the antibodies were raised. Many techniques areavailable for purification and/or concentration of polyclonalantibodies, as well as monoclonal antibodies. See, e.g., Coligan, et al.(current ed.) Unit 9, Current Protocols in Immunology, WileyInterscience.

It is also possible to use the anti-idiotype antibody technology toproduce monoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, e.g., Green, et al. “Production of Polyclonal Antisera”pages 1-5 in Manson (ed.) Immunochemical Protocols Humana Press;Production of Polyclonal Antisera in Rabbits Rats, Mice and Hamsterssection 2.4.1 in Coligan, et al. Current Protocols in Immunology.

The preparation of monoclonal antibodies likewise is typicallyconventional. See, e.g., Kohler and Milstein (1975) Nature 256:495-497;Coligan, et al. Current Protocols sections 2.5.1-2.6.7; and Harlow andLane (1989). Briefly, monoclonal antibodies can be obtained by injectingmice with a composition comprising an antigen, verifying the presence ofantibody production by removing a serum sample, removing the spleen toobtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures. Monoclonal antibodies can be isolated andpurified from hybridoma cultures by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography. See, e.g., Coligan, et al. CurrentProtocols sections 2.7.1-2.7.12 and 2.9.1-2.9.3; Bames, et al. inMethods in Molecular Biology, Humana Press.

Therapeutic applications are conceivable for the antibodies of thepresent invention. For example, antibodies of the present invention mayalso be derived from subhuman primate antibody. General techniques forraising therapeutically useful antibodies in baboons may be found, e.g.,in Goldenberg, et al. (1991) WO 91/11465; and Losman, et al. (1990) Int.J. Cancer 46:310-314.

Alternatively, a therapeutically useful anti-IL-B50 functional receptorantibody may be derived from a “humanized” monoclonal antibody.Humanized monoclonal antibodies are produced by transferring mousecomplementary determining regions from heavy and light variable chainsof the mouse immunoglobulin into a human variable domain, and thensubstituting human residues in the framework regions of the murinecounterparts. The use of antibody components derived from humanizedmonoclonal antibodies obviates potential problems associated with theimmunogenicity of murine constant regions. General techniques forcloning murine immunoglobulin variable domains are described, e.g., byOrlandi, et al. (1989) Proc. Nat'l Acad. Sci. USA 86:3833-3837.Techniques for producing humanized monoclonal antibodies are described,e.g., by Jones, et al. (1986) Nature 321:522-525; Riechmann, et al.(1988) Nature 332:323-327; Verhoeyen, et al. (1988) Science239:1534-1536; Carter, et al. (1992) Proc. Nat'l Acad. Sci. USA89:4285-4289; Sandhu (1992) Crit. Rev. Biotech. 12:437-462; and Singer,et al. (1993) J. Immunol. 150:2844-2857.

Antibodies of the invention also may be derived from human antibodyfragments isolated from a combinatorial immunoglobulin library. See,e.g., Barbas, et al. (1991) Methods: A Companion to Methods inEnzymology, vol. 2, page 119; and Winter, et al. (1994) Ann. Rev.Immunol. 12:433-465. Cloning and expression vectors that are useful forproducing a human immunoglobulin phage library can be obtained, e.g.,from STRATAGENE Cloning Systems (La Jolla, Calif.).

In addition, antibodies of the present invention may be derived from ahuman monoclonal antibody. Such antibodies are obtained from transgenicmice that have been “engineered” to produce specific human antibodies inresponse to antigenic challenge. In this technique, elements of thehuman heavy and light chain loci are introduced into strains of micederived from embryonic stem cell lines that contain targeted disruptionsof the endogenous heavy and light chain loci. The transgenic mice cansynthesize human antibodies specific for human antigens, and the micecan be used to produce human antibody-secreting hybridomas. Methods forobtaining human antibodies from transgenic mice are described by Green,et al. (1994) Nature Genet. 7:13-21; Lonberg, et al. (1994) Nature368:856-859; and Taylor, et al. (1994) Int. Immunol. 6:579-591.

Antibody fragments of the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using papain produces twomonovalent Fab fragments and an Fc fragment directly. These methods aredescribed, e.g., by Goldenberg, U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein. These patents arehereby incorporated in their entireties by reference including allfigures, drawings, and illustrations. See also Nisonhoff, et al. (1960)Arch. Biochem. Biophys. 89:230-244; Porter (1959) Biochem. J.73:119-127; Edelman, et al. (1967) Methods in Enzymology, vol. 1,Academic Press; and Coligan, et al. Current Protocols, at sections2.8.1-2.8.10 and 2.10.1-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent, as described in Inbar, etal. (1972) Proc. Nat'l Acad. Sci. USA 69:2659-2662. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde. See, e.g., Sandhu(1992) Crit. Rev. Biotech. 12:437-462. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, e.g., by Whitlow, et al.(1991) Methods: A Companion to Methods in Enzymology, vol. 2, page 97;Bird, et al. (1988) Science 242:423-426; Ladner, et al., U.S. Pat. No.4,946,778; Pack, et al. (1993) Bio/Technology 11: 1271-77; and Sandhu(1992) Crit. Rev. Biotech. 12:437-462.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, e.g., by usingthe polymerase chain reaction to synthesize the variable region from RNAof antibody-producing cells. See, e.g., Larrick, et al. (1991) Methods:A Companion to Methods in Enzymology, vol. 2, page 106.

Antibodies can be raised to the various mammalian, e.g., IL-B50 receptorcomplex and fragments thereof, both in naturally occurring native formsand in their recombinant forms, the difference being that antibodies tothe active ligand are more likely to recognize epitopes which are onlypresent in the native conformations. Denatured antigen detection canalso be useful in, e.g., Western blot analysis. Anti-idiotypicantibodies are also contemplated, which would be useful as agonists orantagonists of a natural receptor or an antibody.

A number of immunogens may be used to produce antibodies specificallyreactive with receptor complex proteins. Recombinant protein is thepreferred immunogen for the production of monoclonal or polyclonalantibodies. Naturally occurring protein may also be used either in pureor impure form. Synthetic peptides made using the human IL-B50 receptorprotein sequences described herein may also used as an immunogen for theproduction of antibodies to receptor complexes. Recombinant protein canbe expressed in eukaryotic or prokaryotic cells as described herein, andpurified as described. The product is then injected into an animalcapable of producing antibodies. Either monoclonal or polyclonalantibodies may be generated for subsequent use in immunoassays tomeasure the protein.

Methods of producing polyclonal antibodies are known to those of skillin the art. In brief, an immunogen, preferably a purified protein, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to theantigen of interest. When appropriately high titers of antibody to theimmunogen are obtained, blood is collected from the animal and antiseraare prepared. Further fractionation of the antisera to enrich forantibodies reactive to the protein can be done if desired. See Harlowand Lane.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Briefly, spleen cells from an animal immunizedwith a desired antigen are immortalized, commonly by fusion with amyeloma cell. Alternative methods of immortalization includetransformation with Epstein Barr Virus, oncogenes, or retroviruses, orother methods well known in the art. Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, and yield of themonoclonal antibodies produced by such cells may be enhanced by varioustechniques, including injection into the peritoneal cavity of avertebrate host. The genes encoding the derived antibodies may besubjected to mutagenesis to enhance binding affinity or pharmacokineticproperties. Alternatively, one may isolate DNA sequences which encode amonoclonal antibody or a binding fragment thereof by screening a DNAlibrary from human B cells according to the general protocol outlined byHuse, et al. (1989) Science 246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of the protein can be raised byimmunization of animals with conjugates of the fragments withimmunogenic proteins. Monoclonal antibodies are prepared from cellssecreting the desired antibody. These antibodies can be screened forbinding to normal or defective protein, or screened for agonistic orantagonistic activity. These monoclonal antibodies will usually bindwith at least a K_(D) of about 1 mM, more usually at least about 300 μM,typically at least about 100 μM, more typically at least about 30 μM,preferably at least about 10 μM, and more preferably at least about 3 μMor better; including 1 μM, 300 nM, 100 nM, 30 nM, etc.

The antibodies, including antigen binding fragments, of this inventioncan have significant diagnostic or therapeutic value. They can be potentantagonists that bind to the receptor complex and inhibit binding to theligand or inhibit the ability of ligand to elicit a biological response.They also can be useful as non-neutralizing antibodies and can becoupled to toxins or radionuclides to bind producing cells. Further,these antibodies can be conjugated to drugs or other therapeutic agents,either directly or indirectly by means of a linker.

The antibodies of this invention can also be useful in diagnosticapplications. As capture or non-neutralizing antibodies, they can bindto the interleukin without inhibiting receptor binding. As neutralizingantibodies, they can be useful in competitive binding assays. They willalso be useful in detecting or quantifying functional receptor complex.They may be used as reagents for Western blot analysis, or forimmunoprecipitation or immunopurification of the respective protein.

Protein fragments may be joined to other materials, particularlypolypeptides, as fused or covalently joined polypeptides to be used asimmunogens. Primate receptor complex and its fragments may be fused orcovalently linked to a variety of immunogens, such as keyhole limpethemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology,Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962)Specificity of Serological Reactions, Dover Publications, New York; andWilliams, et al. (1967) Methods in Immunology and Immunochemistry, Vol.1, Academic Press, New York; each of which are incorporated herein byreference, for descriptions of methods of preparing polyclonal antisera.A typical method involves hyperimmunization of an animal with anantigen. The blood of the animal is then collected shortly after therepeated immunizations and the gamma globulin is isolated.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.), Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratorv Manual,CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York; and particularly in Kohler andMilstein (1975) in Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Each of these references isincorporated herein by reference. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides or alternatively to selection of libraries ofantibodies in phage or similar vectors. See, Huse, et al. (1989)“Generation of a Large Combinatorial Library of the ImmunoglobulinRepertoire in Phage Lambda,” Science 246:1275-1281; and Ward, et al.(1989) Nature 341:544-546, each of which is hereby incorporated hereinby reference. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant or chimeric immunoglobulins may beproduced, see Cabilly, U.S. Pat. No. 4,816,567; or made in transgenicmice, see Mendez, et al. (1997) Nature Genetics 15:146-156. Thesereferences are incorporated herein by reference.

The antibodies of this invention can also be used for affinitychromatography in isolating primate receptor or cells expressing such.Columns can be prepared where the antibodies are linked to a solidsupport, e.g., particles, such as agarose, SEPHADEX, or the like, wherea cell lysate may be passed through the column, the column washed,followed by increasing concentrations of a mild denaturant, whereby thepurified protein will be released. Conversely, protein may be used topurify antibody.

Antibodies may also be used to screen expression libraries forparticular expression products. Usually antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies raised against an IL-B50 receptor will also be used to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of theprotein or cells which express receptors for the protein. They also willbe useful as agonists or antagonists of the IL-B50, which may becompetitive inhibitors or substitutes for naturally occurring ligands.

Binding Composition:Receptor Protein Complex

An IL-B50 receptor that specifically binds to or that is specificallyimmunoreactive with an antibody, e.g., a polyclonal antibody generatedagainst a defined immunogen, e.g., an immunogen consisting of a complexcomprising both IL-7Rα and Rδ2, is typically determined in animmunoassay. Included within the present invention are those nucleicacid sequences described herein, including functional variants, thatencode polypeptides that combine to bind to polyclonal antibodiesgenerated against the prototypical primate IL-B50 receptor, but not theprior known individual components. The immunoassay typically uses apolyclonal antiserum which was raised, e.g., to a complex comprisingproteins of SEQ ID NO: 2 and 4. This antiserum is selected to have lowcrossreactivity against other IL-7 receptor family members, preferablyfrom the same species, and to other rodent or similar evolutionarilydistant receptors, so that any such crossreactivity is removed byimmunoabsorption prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the functionalcomplex is isolated as described herein. For example, recombinantprotein may be produced in a mammalian cell line. An appropriate host,e.g., an inbred strain of mice such as Balb/c, is immunized with thecomplex using a standard adjuvant, such as Freund's adjuvant, and astandard mouse immunization protocol (see Harlow and Lane).Alternatively, a synthetic peptide derived from the sequences disclosedherein and conjugated to a carrier protein can be used as an immunogen.Polyclonal sera are collected, perhaps immunodepleted, and titeredagainst the immunogen protein in an immunoassay, e.g., a solid phaseimmunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10⁴ or greater are selected andtested for their cross reactivity against other IL-7 receptor familymembers, e.g., using a competitive binding immunoassay such as the onedescribed in Harlow and Lane, supra, at pages 570-573. Preferably atleast two IL-7 receptor family members are used in this determination.These IL-7 receptor family members can be produced as recombinantproteins and isolated using standard molecular biology and proteinchemistry techniques as described herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, the receptor complex,soluble or membrane associated, can be immobilized to a solid support.Proteins added to the assay compete with the binding of the antisera tothe immobilized antigen. The ability of the above proteins to competewith the binding of the antisera to the immobilized complex is comparedto the other family members. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorption with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second proteincomplex to the immunogen protein complex. In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required is less than twice the amountof the antigen receptor complex that is required, then the secondprotein is said to specifically bind to an antibody generated to theimmunogen.

VII. Kits and Quantitation

Both naturally occurring and recombinant forms of the receptor complexof this invention are particularly useful in kits and assay methods. Forexample, these methods would also be applied to screening for bindingactivity, e.g., ligands or antagonists for these receptors. Severalmethods of automating assays have been developed in recent years so asto permit screening of tens of thousands of compounds per year. See,e.g., a BIOMEK automated workstation, Beckman Instruments, Palo Alto,Calif., and Fodor, et al. (1991) Science 251:767-773, which isincorporated herein by reference. The latter describes means for testingbinding by a plurality of defined polymers synthesized on a solidsubstrate. The development of suitable assays to screen for anagonist/antagonist or ligand-like proteins can be greatly facilitated bythe availability of large amounts of purified, soluble receptorcomplexes in an active state such as is provided by this invention.

Purified receptor complexes can be attached to substrates for use in theaforementioned screening techniques. However, non-neutralizingantibodies to these receptors can be used as capture antibodies toimmobilize the respective receptor complexes on the solid phase, useful,e.g., in diagnostic uses.

This invention also contemplates use of receptor complexes, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of the receptor.Alternatively, or additionally, antibodies against the molecules may beincorporated into the kits and methods. Typically the kit will have acompartment containing either a defined peptide or gene segment or areagent which recognizes one or the other. Typically, recognitionreagents, in the case of peptide, would be a ligand or antibody, or inthe case of a gene segment, would usually be a hybridization probe.

A preferred kit for determining the concentration of, e.g., receptorcomplex, a sample would typically comprise a labeled compound, e.g.,IL-B50 or antibody, having known binding affinity for receptor, a sourceof receptor (naturally occurring or recombinant) as a positive control,and a means for separating the bound from free labeled compound, e.g., asolid phase for immobilizing the receptor in the test sample.Compartments containing reagents, and instructions, will normally beprovided.

Antibodies, including antigen binding fragments, specific for mammalianreceptor complex or a peptide fragment, or ligand are useful indiagnostic applications to detect the presence of elevated levels ofreceptor and/or its fragments. Diagnostic assays may be homogeneous(without a separation step between free reagent and antibody-antigencomplex) or heterogeneous (with a separation step). Various commercialassays exist, such as radioimmunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multipliedimmunoassay technique (EMIT), substrate-labeled fluorescent immunoassay(SLFIA) and the like. For example, unlabeled antibodies can be employedby using a second antibody which is labeled and which recognizes theantibody to receptor complex or to a particular fragment thereof. Theseassays have also been extensively discussed in the literature. See,e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH., andColigan (ed. 1991 and periodic supplements) Current Protocols InImmunology Greene/Wiley, New York.

Anti-idiotypic antibodies may have similar use to serve as agonists orantagonists of receptor. These should be useful as therapeutic reagentsunder appropriate circumstances.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody, or labeled ligand is provided.This is usually in conjunction with other additives, such as buffers,stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent, andwill contain instructions for proper use and disposal of reagents.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium havingappropriate concentrations for performing the assay.

Many of the aforementioned constituents of the diagnostic assays may beused without modification or may be modified in a variety of ways. Forexample, labeling may be achieved by covalently or non-covalentlyjoining a moiety which directly or indirectly provides a detectablesignal. In any of these assays, a test compound, receptor complex, orantibodies thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups: radiolabels suchas ¹²⁵I, enzymes (U.S. Pat. No. 3,645,090) such as peroxidase andalkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475)capable of monitoring the change in fluorescence intensity, wavelengthshift, or fluorescence polarization. Both patents are incorporatedherein by reference. Possibilities for indirect labeling includebiotinylation of one constituent followed by binding to avidin coupledto one of the above label groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. Thereceptor complex can be immobilized on various matrixes followed bywashing. Suitable matrices include plastic such as an ELISA plate,filters, and beads. Methods of immobilizing the receptor to a matrixinclude, without limitation, direct adhesion to plastic, use of acapture antibody, chemical coupling, and biotin-avidin. The last step inthis approach involves the precipitation of antibody/antigen complex byany of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30(9):1457-1461, and the double antibodymagnetic particle separation as described in U.S. Pat. No. 4,659,678,each of which is incorporated herein by reference.

The methods for linking protein or fragments to various labels have beenextensively reported in the literature and do not require detaileddiscussion here. Many of the techniques involve the use of activatedcarboxyl groups either through the use of carbodiimide or active estersto form peptide bonds, the formation of thioethers by reaction of amercapto group with an activated halogen such as chloroacetyl, or anactivated olefin such as maleimide, for linkage, or the like. Fusionproteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequences ofthese primate receptor subunits. These sequences can be used as probesfor detecting levels of the receptors in patients suspected of having animmunological disorder, or to evaluate polymorphic variation. Thepreparation of both RNA and DNA nucleotide sequences, the labeling ofthe sequences, and the preferred size of the sequences has receivedample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²p. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorescers, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out in any conventional techniques such as nucleicacid hybridization, plus and minus screening, recombinational probing,hybrid released translation (HRT), and hybrid arrested translation(HART). This also includes amplification techniques such as polymerasechain reaction (PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

VIII. Therapeutic Utility

This invention provides reagents with significant therapeutic value. Thereceptor (naturally occurring or recombinant), fragments thereof,soluble constructs, and antibodies, along with compounds identified ashaving binding affinity to the receptor, are useful in the treatment ofconditions exhibiting abnormal expression of the cytokine or receptor.Such abnormality will typically be manifested by immunological disordersor otherwise, as described. Additionally, this invention providestherapeutic value in various diseases or disorders associated withabnormal expression or abnormal triggering of response to theinterleukin.

In addition, the expression profiles of functional receptor subunitssuggests what cells should be responsive to the ligand, e.g., IL-B50. Inparticular, cells of the lymphoid lineage, e.g., macrophages anddendritic cells, express both subunits in sufficient stoichiometricamounts to form functional complexes. In particular, since the IL-7Rα isshared with the IL-7 ligand, it would be expected that much of thesignaling pathway will be very similar to IL-7. Thus, much of thebiology of the same cells will be closely related. In contrast, theexpression of the IL-B50 receptor subunits in different cell typessuggests that signaling to those cell types occurs, and will haveeffects on the physiology effected by such cell types. An antagonist,mutein or antibody, could prove very useful in those situations. SeeRich (ed.) Clinical Immunology: Principles and Practice, Mosby.

T helper cells mediate effector functions in infectious, allergic, orautoimmune diseases through production of cytokines. CD4+ T cells can bedivided into Th1 and Th2 subsets on the basis of their cytokine profileupon antigen stimulation. Evidence has recently been obtained that Th1and Th2 cells differ in responsiveness and receptor expression. Thelinkage via TARC expression in response to IL-B50, and production of Th2type cytokines suggests effect on Th2 responses. The expression profileof the proteins here described indicates that IL-B50 is the ligand forfunctional receptor complex and, as such, could be important for Th2effector functions.

Recombinant soluble receptor may be useful as an antagonist, antibodiesagainst the receptor subunits or complex, or IL-B50 antibodies orcytokine muteins can be purified and then administered to a patient.These reagents can be combined for therapeutic use with additionalactive ingredients, e.g., in conventional pharmaceutically acceptablecarriers or diluents, along with physiologically innocuous stabilizersand excipients. These combinations can be sterile, e.g., filtered, andplaced into dosage forms as by lyophilization in dosage vials or storagein stabilized aqueous preparations. This invention also contemplates useof antibodies or binding fragments thereof which are not complementbinding.

Further analysis can be performed to identify additional moleculesinvolved in the receptor to the cytokine, e.g., additional receptorsubunits. Subsequent biological assays can then be utilized to determineif those additional receptor components can affect signal transduction,which can block provide means to further address mechanisms orstructures in the signaling pathways. Receptor fragments, e.g., solublereceptor constructs, or ligand muteins can be used as a blocker orantagonist which blocks the activity of IL-B50. Conversely, a compoundhaving intrinsic stimulating activity can activate the receptor and isthus an agonist in that it simulates the activity of IL-B50. Thisinvention further contemplates the therapeutic use of antibodies to thereceptor as agonists.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics,8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences,(current ed.), Mack Publishing Co., Easton, Pa.; each of which is herebyincorporated herein by reference. Methods for administration arediscussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or slow release apparatus will often be utilized forcontinuous administration.

Receptor subunits, complexes of subunits, fragments thereof, andantibodies or their fragments, antagonists, and agonists, or vectorsencoding any of the mentioned entities may be administered directly tothe host to be treated or, depending on the size of the compounds, itmay be desirable to conjugate them to carrier proteins such as ovalbuminor serum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationscomprise at least one active ingredient, as defined above, together withone or more acceptable carriers thereof. Each carrier must be bothpharmaceutically and physiologically acceptable in the sense of beingcompatible with the other ingredients and not injurious to the patient.Formulations include those suitable for oral, rectal, nasal, orparenteral (including subcutaneous, intramuscular, intravenous andintradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8thEd., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed.(1990), Mack Publishing Co., Easton, Pa.; Avis, et al. (eds. 1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: TabletsDekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY.

Another therapeutic approach included within the invention involvesdirect administration of reagents or compositions by any conventionaladministration techniques (e.g., but not restricted to local injection,inhalation, or administered systemically), to the subject with anappropriate medical disorder. The reagent, formulation, or compositionmay also be targeted to specific cells or receptors by methods describedherein. The actual dosage of reagent, formulation or composition thatmodulates an inflammatory disorder depends on many factors, includingthe size and health of an organism. See, e.g., Spilker (1984) Guide toClinical Studies and Developing Protocols, Raven Press, New York,particularly pages 7-13, 54-60; Spilker (1991) Guide to Clinical Trials,Raven Press, New York, especially pages 93-101; Craig and Stitzel (eds.1986) Modern Pharmacology 2d ed., Little, Brown, Boston, especiallypages 127-33; Speight (ed. 1987) Avery's Drug Treatment: Principles andPractice of Clinical Pharmacology and Therapeutics, 3d ed., Williams andWilkins, Baltimore, pages 50-56; and Tallarida, et al. (1988) Principlesin General Pharmacology, Springer-Verlag, New York, pages 18-20; whichdescribes how to determine the appropriate dosage. Generally, doses inthe range of between about 0.5 ng/ml and 500 μg/ml inclusive finalconcentration are administered per day to an adult in apharmaceutically-acceptable carrier. The therapy of this invention maybe combined with or used in association with other therapeutic agentsdirected to the indicated conditions, particularly agonists orantagonists of other IL-7 family members.

The IL-B50 receptor complex forms the basis for antagonist drugdevelopment (e.g., humanized anti-human receptor antibodies). IL-B50 mayform an integral part of the lymphoid lineage immune defense. The IL-B50appears to signal through the STAT3 and STAT5 molecule pathways.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation. It is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by preferred embodiments and optionalfeatures, modification and variation of the concepts herein disclosedmay be used by those of ordinary skill in the art, and that suchmodifications and variations are considered to be within the scope ofthis invention as defined by the appended claims.

EXAMPLES

I. General Methods

Some of the standard methods are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual, (2d ed.), vols 1-3, CSHPress, NY; Ausubel, et al., Biology, Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology, Greene/Wiley, New York. Methods forprotein purification include such methods as ammonium sulfateprecipitation, column chromatography, electrophoresis, centrifugation,crystallization, and others. See, e.g., Ausubel, et al. (1987 andperiodic supplements); Deutscher (1990) “Guide to Protein Purification”in Meth. Enzymol., vol. 182, and other volumes in this series; andmanufacturer's literature on use of protein purification products, e.g.,Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, Calif. Combinationwith recombinant techniques allow fusion to appropriate segments, e.g.,to a FLAG sequence or an equivalent which can be fused via aprotease-removable sequence. See, e.g., Hochuli (1989) ChemischeIndustrie 12:69-70; Hochuli (1990) “Purification of Recombinant Proteinswith Metal Chelate Absorbent” in Setlow (ed.) Genetic Engineering,Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al.(1992) QIAexpress: The High Level Expression & Protein PurificationSystem QIAGEN, Inc., Chatsworth, Calif.

Computer sequence analysis is performed, e.g., using available softwareprograms, including those from the GCG (U. Wisconsin) and GenBanksources. Public sequence databases were also used, e.g., from GenBankand others.

Many techniques applicable to the IL-10 receptor may be applied toIL-7Rα and/or Rδ2, as described, e.g., in U.S. Pat. No. 5,985,828, whichis incorporated herein by reference for all purposes.

II. Amplification of Receptor Fragments by PCR

Cloning of human receptors is performed by standard procedures. Variousmethods of amplifying target sequences, such as the polymerase chainreaction, can also be used to prepare DNA encoding these receptorproteins or polypeptides. Polymerase chain reaction (PCR) technology isused to amplify such nucleic acid sequences directly from mRNA, fromcDNA, and from genomic libraries or cDNA libraries. This allows fordiagnostic methods which allow determination of polymorphic orpopulational variety, may of which might affect physiology or functionof the resulting gene product. Such may allow predictive information tobe gathered to diagnose disease or predict therapeutic response.

In PCR techniques, oligonucleotide primers complementary to two 5′regions in the DNA region to be amplified are synthesized. Thepolymerase chain reaction is then carried out using the two primers. SeeInnis et al. (current eds.) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif. Primers can be selectedto amplify the entire regions encoding full-length receptor proteins orto amplify smaller DNA segments as desired. Once such regions arePCR-amplified, they can be sequenced and oligonucleotide probes can beprepared from sequence obtained using standard techniques.

Oligonucleotides for use as probes are chemically synthesized accordingto the solid phase phosphoramidite triester method first described byBeaucage and Carruthers (1983) Tetrahedron Lett. 22(20): 1859-1862, orusing an automated synthesizer, as described in Needham-VanDevanter etal. (1984) Nucleic Acids Res. 12: 6159-6168. Purification ofoligonucleotides is performed, e.g., by native acrylamide gelelectrophoresis or by anion-exchange HPLC as described in Pearson andRegnier (1983) J. Chrom. 255: 137-149. The sequence of the syntheticoligonucleotide can be verified using the chemical degradation method ofMaxam and Gilbert in Grossman and Moldave (eds. 1980) Methods inEnzymology 65:499-560 Academic Press, New York.

The peptide segments, along with comparison to homologous genes, canalso be used to produce appropriate oligonucleotides to screen alibrary. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library

Complementary sequences will also be used as probes or primers. Basedupon identification of the likely amino terminus, other peptides shouldbe particularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides.

To identify a homologous receptor protein, degenerate oligonucleotidesare designed. The primers are used for polymerase chain reactions on,e.g., genomic DNA followed by subcloning the PCR products usingrestriction sites placed at the 5′ ends of the primers, pickingindividual E. coli colonies carrying these subcloned inserts, and usinga combination of random sequencing and hybridization analysis toeliminate other known family members.

Subsequently, PCR products are gel-purified, digested with appropriaterestriction enzymes, gel-purified again, and subcloned in the Bluescriptvector (Stratagene, San Diego, Calif.). Bacterial colonies carryingindividual subclones are picked into 96 well microtiter plates, andmultiple replicas are prepared by plating the cells onto nitrocellulose.The replicate filters are hybridized to probes representing knownmembers of the IL-7 receptor family, and DNA is prepared fromnon-hybridizing colonies for sequence analysis.

Two appropriate forward and reverse primers are selected using thesequences supplied herein (see FIGS. 1 and 2) and common knowledge. See,e.g., Innis, et al. (current eds.) PCR Protocols: A Guide to Methods andApplications Academic Press, San Diego, Calif.; and Dieffenbach andDveksler (current eds.) PCR Primer: A Laboratory Manual Cold SpringHarbor Press, CSH, NY. RT-PCR is used on an appropriate mRNA sampleselected for the presence of message to produce a cDNA, e.g., a monocyteor macrophage cell sample.

Full length clones may be isolated by hybridization of cDNA librariesfrom appropriate tissues pre-selected by PCR signal.

As is commonly known, PCR primers are typically designed to contain atleast 15 nucleotides, e.g., 15-30 nucleotides. The design of specificprimers containing 21 nucleotides, e.g., that code for the appropriatepolypeptides containing at least 4 amino acids from the IL7Rα or Rδ2sequences are described as follows. Other PCR primers designed toamplify other receptor polypeptide fragments will be designed in asimilar fashion, e.g., mutagenesis primers. Preferably, most or all ofthe nucleotides in such a primer encode conserved amino acids, e.g.,amino acid residues of SEQ ID NO: 2 or 4. For example, primerscontaining at least 40% IL-7Rα or Rδ2 conserved amino acids can be used.Once appropriate amino acids are selected as templates against whichprimer sequences are to be designed, the primers can be synthesizedusing, e.g., standard chemical methods. Due to the degeneracy of thegenetic code and the bias of preferred species variants, such primersshould be designed to include appropriate degenerate sequences, as canbe readily determined using common knowledge.

III. Tissue distribution of IL-7Rα or Rδ2

Message for the gene encoding IL-7Rα has been detected in a human cDNAlibrary, in dendritic cells and monocytes. Message for Rδ2 has beendetected in dendritic cells, monocytes, NK, and some T cells. Both arefound in certain DC and certain monocyte samples, indicating that thecomponents of functional receptors are on those cells.

Southern Analysis: DNA (5 μg) from a primary amplified cDNA library isdigested with appropriate restriction enzymes to release the inserts,run on a 1% agarose gel and transferred to a nylon membrane (Schleicherand Schuell, Keene, N.H.).

Samples for human mRNA isolation could include: peripheral bloodmononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells),resting (T100); peripheral blood mononuclear cells, activated withanti-CD3 for 2, 6, 12 h pooled (T101); T cell, TH0 clone Mot 72, resting(T102); T cell, TH0 clone Mot 72, activated with anti-CD28 and anti-CD3for 3, 6, 12 h pooled (T103); T cell, TH0 clone Mot 72, anergic treatedwith specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 cloneHY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06,anergic treated with specific peptide for 2, 6, 12 h pooled (T109); Tcell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935,activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T 111); Tcells CD4+ CD45RO− T cells polarized 27 days in anti-CD28, IL-4, andanti IFN-γ, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h(T116); T cell tumor lines Jurkat and Hut78, resting (T117); T cellclones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting(T118); T cell random γδ T cell clones, resting (T119); Splenocytes,resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); Bcell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting(B102); B cell line JY, activated with PMA and ionomycin for 1, 6 hpooled (B103); NK 20 clones pooled, resting (K100); NK 20 clones pooled,activated with PMA and ionomycin for 6 h (K101); NKL clone, derived fromperipheral blood of LGL leukemia patient, IL-2 treated (K106); NKcytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor lineTF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937premonocytic line, resting (M100); U937 premonocytic line, activatedwith PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes,activated with LPS, IFNγ, anti-IL-10 for 1, 2, 6, 12, 24 h pooled(M102); elutriated monocytes, activated with LPS, IFNγ, IL-10 for 1, 2,6, 12, 24 h pooled (M103); elutriated monocytes, activated with LPS,IFNγ, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes,activated with LPS, IFNγ, IL-10 for 4, 16 h pooled (M107); elutriatedmonocytes, activated LPS for 1 h (M108); elutriated monocytes, activatedLPS for 6 h (M109); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days,resting (D101); DC 70% CD1a+, from CD34+ GM-CSF, TNFα 12 days, activatedwith PMA and ionomycin for 1 hr (D102); DC 70% CD1a+, from CD34+ GM-CSF,TNFα12 days, activated with PMA and ionomycin for 6 hr (D103); DC 95%CD1a+, from CD34+ GM-CSF, TNFα 12 days FACS sorted, activated with PMAand ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF,TNFα 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hrpooled (D105); DC CD1a+ CD86+, from CD34+ GM-CSF, TNFα 12 days FACSsorted, activated with PMA and ionomycin for 1, 6 h pooled (D106); DCfrom monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytesGM-CSF, IL-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF,IL-4 5 days, activated TNFα, monocyte supe for 4, 16 h pooled (D110);leiomyoma L11 benign tumor (X101); normal myometrium M5 (O115);malignant leiomyosarcoma GS1 (X103); lung fibroblast sarcoma line MRC5,activated with PMA and ionomycin for 1, 6 h pooled (C101); kidneyepithelial carcinoma cell line CHA, activated with PMA and ionomycin for1, 6 h pooled (C102); kidney fetal 28 wk male (O100); lung fetal 28 wkmale (O101); liver fetal 28 wk male (O102); heart fetal 28 wk male(O103); brain fetal 28 wk male (O104); gallbladder fetal 28 wk male(O106); small intestine fetal 28 wk male (O107); adipose tissue fetal 28wk male (O108); ovary fetal 25 wk female (O109); uterus fetal 25 wkfemale (O110); testes fetal 28 wk male (O111); spleen fetal 28 wk male(O112); adult placenta 28 wk (O113); and tonsil inflamed, from 12 yearold (X100).

IV. Production of Mammalian Receptor Protein

Typically, co-expression will be useful, and constructs can be made toproduce soluble receptors. This may be effected by a polycistronicconstruct in appropriate cells, or by construction of vectors whichcoexpress both receptor subunits together. Alternatively, fusionconstructs can be produced, e.g., to generate antibodies.

An appropriate construct is engineered for expression, e.g., in E. coli.For example, a mouse IGIF pGex plasmid is constructed and transformedinto E. coli. Freshly transformed cells are grown in LB mediumcontaining 50 μg/ml ampicillin and induced with IPTG (Sigma, St. Louis,Mo.). After overnight induction, the bacteria are harvested and thepellets containing receptors are isolated. The pellets are homogenizedin TE buffer (50 mM Tris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2liters. This material is passed through a microfluidizer (Microfluidics,Newton, Mass.) three times. The fluidized supernatant is spun down on aSorvall GS-3 rotor for 1 h at 13,000 rpm. The resulting supernatantcontaining the receptor is filtered and passed over aglutathione-SEPHAROSE column equilibrated in 50 mM Tris-base pH 8.0. Thefractions containing the receptor protein are pooled and cleaved withthrombin (Enzyme Research Laboratories, Inc., South Bend, Ind.). Thecleaved pool is then passed over a Q-SEPHAROSE column equilibrated in 50mM Tris-base. Fractions containing receptor are pooled and diluted incold distilled H₂O, to lower the conductivity, and passed back over afresh Q-SEPHAROSE column. Fractions containing receptor are pooled,aliquoted, and stored in the −70 degrees C. freezer.

Comparison of the CD spectrum with functional receptor may suggest thatthe protein is correctly folded. See Hazuda, et al. (1969) J. Biol.Chem. 264:1689-1693.

Protein expression and purification of human receptor: Adenoviralvectors containing full-length human receptors are constructed by PCRand used to transfect, e.g., Q293 packaging cells. Produced viruses aresubsequently purified, with all procedures according to manufacturer'sprotocols (Invitrogen). Receptor proteins are prepared, e.g., from 5×10⁸infected Q293 cells (adenoviruses at 10 MOI) which are subsequentlyincubated for 5 days in a cell factory in a total volume of 11 ofserum-free CMF-1 medium (Gibco BRL). Culture medium is dialyzed(Spectra/Por membrane tubing, MW cut off: 6-8 kD) against 50 mMTris-HCl, pH 8.0, 1 mM EDTA, and subsequently passed over Hitrap Qsepharose and Heparin columns. The flow-through, containing the receptorproteins, is sterile-filtered and concentrated approximately 70 timesusing an Amicon 8400 ultrafiltration cell with a 10 kD MW cut offmembrane. The samples are dialyzed against PBS, and the protein contentquantified by PAGE and Coomassie Blue staining using lysozyme as astandard. Protein identities are confirmed, e.g., by N-terminalsequencing. Identically treated Q293 cells infected with adenovirusencoding green fluorescent protein provide a mock control. Endotoxinlevels are determined using the Limulus Amebocyte Lysate assay(BioWhittaker) and were less then 4 EU/ml. Protein samples are stored at4 degrees C.

Expression vectors: For mammmalian expression, vectors encodingfull-length human receptor are constructed by inserting PCR-generatedcDNA fragments into pME18S. Kitamura, et al. (1991) Proc. Nat'l Acad.Sci. USA 88:5082-5086.

V. Preparation of Antibodies Specific for Receptor Complex

Inbred Balb/c mice are immunized intraperitoneally with recombinantforms of the protein, e.g., purified soluble receptor-FLAG or stabletransfected NIH-3T3 cells. Animals are boosted at appropriate timepoints with protein or cells, with or without additional adjuvant, tofurther stimulate antibody production. Serum is collected, or hybridomasproduced with harvested spleens.

Alternatively, Balb/c mice are immunized with cells transformed with thegene or fragments thereof, either endogenous or exogenous cells, or withisolated membranes enriched for expression of the antigen. Serum iscollected at the appropriate time, typically after numerous furtheradministrations. Various gene therapy techniques may be useful, e.g., inproducing protein in situ, for generating an immune response.

Monoclonal antibodies may be made. For example, splenocytes are fusedwith an appropriate fusion partner and hybridomas are selected in growthmedium by standard procedures. Hybridoma supernatants are screened forthe presence of antibodies which bind to the desired receptor, e.g., byELISA or other assay. Antibodies which specifically recognize receptoror complex may also be selected or prepared.

In another method, synthetic peptides or purified protein are presentedto an immune system to generate monoclonal or polyclonal antibodies.See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press. In appropriate situations, the binding reagent is eitherlabeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. Nucleic acids may alsobe introduced into cells in an animal to produce the antigen, whichserves to elicit an immune response. See, e.g., Wang, et al. (1993)Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniques16:616-619; and Xiang, et al. (1995) Immunity 2: 129-135.

VI. Biological Activity of IL-B50 and Complexes with IL-7Rα and Rδ2

A number of experiments were conducted in order to assess the signalingreceptor complex for IL-B50, as well as the biological function ofIL-B50. In the following examples, the term “human TSLP” or “hTSLP” isused interchangeably with the term IL-B50. Additionally, the term “humanTSLPR” or “hTSLPR” is used interchangeably with the term Rδ2. Materialsand methods used in the following experiements were as follows.

Cell lines. Human 293T epithelial cells were maintained in Dulbecco'smodified Eagle's medium (DMEM) (Life Technologies Inc.) supplementedwith 10% fetal calf serum (FCS) (LTBMC). The Pro-B cell line Ba/F3 wasmaintained in RPMI 1640 medium (Life Technologies Inc.) supplementedwith 10% fetal calf serum and 10 ng/ml of mouse IL-3. QBI-293A humanembryonic kidney cells used for adenovirus expression were grown inCMF-1 medium (CellWorks, San Diego, Calif.). BOSC23 cells weremaintained in DMEM-10% FCS and guanine phosphoribosyltransferase (GPT)selection reagents (Specialty Media). The cells were transferred toDulbecco's modified Eagle's medium-10% FCS without GPT selectionreagents 2 days before transfection.

Adenovirus expression of human TSLP (IL-B50) and purification of therecombinant protein. The mature coding region of human TSLP (residues1-131 of FIG. 3) was fused to the signal sequence of mouse SLAM (Bates,et al. (1999) J. Immunol. 163:1973) and inserted into a modified versionof transfer vector pQB1-AdCMV5-GFP (Quantum Biotechnologies Inc.) byPCR. Recombinant adenovirus was produced as described in Quantumapplications manual 24AL98. Recombinant virus was used to infect 5×10⁸cells in 1 L CMF-1 with culture in a Nunc Cell Factory (Nalge Nunc Int.,Naperville, Ill.) for 3 days. The culture medium was clarified bycentrifugation, dialyzed and filtered prior to application to a 5 mlQ-Sepharose column. The Q-Sepharose flow-through, which contained humanTSLP, was loaded onto a 5 ml HiTrap Heparin (Pharmacia, Uppsala, Sweden)column at 5 ml/min. The column was washed with 50 mM Tris-HCl pH 8.0, 1mM EDTA, and eluted with a gradient from 0-2.5 M NaCl in 50 mM Tris-HClpH 8.0, 1 mM EDTA. The peak fractions were concentrated, dialyzedagainst PBS and quantitated by SDS-PAGE and Coomassie staining usinglysozyme as a standard. A similar procedure was followed to preparemouse TSLP.

Ba/F3 retroviral-mediated gene transfer and proliferation assays. HumanIL-7Rα cDNA and human TSLPR cDNA were cloned by PCR in the retroviralvectors pMX and its derivative pMX-puro to give pMX-hIL-7Rα andpMX-puro-TSLPR, respectively (Kitamura, T. (1998) Int. J. Hematol.67:351). The BOSC23 packaging cell line was transiently transfected withretrovirus constructs using Fugene 6 (GIBCO BRL) according to themanufacturer protocol. Retrovirus containing supernatants were collectedafter two days. Ba/F3 cells were infected with retroviral supernatantsfor 48 hr on petri dishes coated with 40 μg/ml recombinant fibronectinfragments (Retronectin, Takara). After 48 hr puromycine (1 μg/ml) wasadded to those cells infected with virus obtained from pMX-puroconstructs. The efficiency of infection of Ba/F3 cells was over 90% asassessed by parallel infection with the test construct pMXI-EGFPencoding the enhanced green fluorescent protein (EGFP). Proliferationassays using Ba/F cells were as previously described (Ho, et al. (1993)Proc. Natl. Acad. Sci. USA 90:11267). Cells were washed three times withRPMI media and plated at a density of 5000 cells/well. Cells were grownwith serial threefold dilutions of mouse IL-3, human and mouse TSLP, orhuman IL-7 (all starting concentrations 225 ng/ml). After 36 hr at 37degrees C. Alamar Blue® REDOX indicator (Trek Diagnostic systems) wasadded to a final concentration of 10% (vol/vol) to each well. Cells wereallowed to grow for 5-8 more hours after which plates were measured witha fluorometer.

Quantitation of mRNA expression. cDNA libraries from various tissues andcellular sources were prepared as described previously (Bolin, et al.(1997) J. Neurosci. 17:5493) and used as templates for Taqman-PCRanalyses. cDNAs (50 ng per reaction) were analyzed for the expression ofhTSLP, hTSLPR and hIL7Rα genes by the Fluorogenic 5′-nuclease PCR assay(Holland, et al. (1991) Proc. Natl. Acad. Sci. USA 88:7276), using anABI Prism 7700 Sequence Detection System (Perkin Elmer, Foster City,Calif.). Reactions were incubated for 2 min at 50 degrees C., denaturedfor 10 min at 95 degrees C. and subjected to 40 two-step amplificationcycles with annealing/extension at 60 degrees C. for 1 min followed bydenaturation at 95 degrees C. for 15 sec. The amplicons used for hTSLP,hTSLPR and IL-7Rα covered bp 246-315, bp 263-335 and bp 519-596,respectively (numbering starts at start codon), and were analyzed withFAM-labeled probes. Values were expressed as fg/50 ng total cDNA.Primers and probes for human chemokine and chemokine receptors wereobtained from Perkin-Elmer as PreDeveloped Assay Reagents (PDAR's).Chemokine and chemokine receptor expression was adjusted for the amountof 18SrRNA and compared to the control (calibrator) samples using thecomparative C_(T) method (Fehniger, et al. (1999) J. Immunol. 162:4511).Samples were measured in duplicate. 18SrRNA levels were determined underprimer limited conditions in multiplex reactions as recommended using aVic labelled probe (Perkin Elmer, Foster City, Calif.).

Cell isolation and culture. Peripheral Blood Mononuclear Cells (PBMC)were purified from buffy coats of healthy volunteers (Stanford BloodBank, Palo Alto, Calif.) by centrifugation over Ficoll. Human monocyteswere isolated from PBMC by negative depletion using anti-CD2 (Leu-5A),anti-CD3 (Leu-4), anti-CD8 (Leu 2a), anti-CD19 (Leu-12), anti-CD20(Leu-16), anti-CD56 (Leu-19), (BD, San Jose Calif.), anti-CD67 (IOM 67)(Immunotech, Westbrook, Me.) and anti-glycophorin A (10F7MN, ATCC,Rockville, Md.) mAbs and sheep anti-mouse IgG coupled magnetic beads(Dynal, Oslo, Norway) as described previously (Koppelman, et al. (1997)Immunity 7:861). Monocytes were cultured in RPMI+ 10% FCS at a densityof 10⁶ cells/ml in the presence or absence of IL-7 (50 ng/ml) and/orhTSLP (50 ng/ml) for 24 hrs and culture supernatants and cells wereharvested for quantitation of cytokine production or gene expressionanalyses. Human CD11c+ dendritic cells (DC) were isolated from PBMC aspreviously described (Kadowaki, et al. (2000) J. Exp. Med. 192:219).Briefly, PBMC were incubated with anti-CD3, anti-CD14, anti-CD19,anti-CD56 mAbs, depleted from lineage+ cells using magnetic beads(Dynal) and CD11c+ Lineage-blood DC were subsequently isolated by cellsorting to reach a purity of more than 99%. Freshly sorted cells werecultured in RPMI1640 containing 10% FCS at 5×10⁴/100 μl in flat-bottom96-well half-area culture plates or at 1×10⁵/200 μl in flat-bottom96-well plates, with or without IL-B50 (15 ng/ml).

TARC Elisa. The production of TARC/CCL17 in culture supernatants wasdetermined by chemokine specific elisa using MAB364 as capture reagentand BAF364 as detection reagent (R&D Systems, Minneapolis Minn.). Thesensitivity of the assay was 50 pg/ml.

DC viability and flow cytometric analysis. After 24 hours of culture, DCwere harvested and resuspended in an EDTA-containing medium todissociate the clusters. Viable DC were first counted using trypan blueexclusion of dead cells. Remaining cells were stained with a variety ofmouse anti-human FITC-conjugated monoclonal antibodies (mAb) includinganti-HLA-DR (Becton Dickinson), anti-CD40, anti-CD80 and CD86 (all fromPharmingen) or an Ig-G1 isotype control (Becton Dickinson), and wereanalyzed with a FACScan® flow cytometer (Becton Dickinson). Dead cellswere excluded based on side and forward scatter characteristics.

T cell proliferation assay. Naïve CD4+/CD45RA+ T cells were isolatedfrom adult blood buffy coats by negative depletion of cells expressingCD14, CD19, CD56, CD8, CD45RO, HLA-DR and glycophorin A using magneticbeads (Dynal). More than 95% of the purified cells had the CD4+CD45RA+naïve T cell phenotype. CD11c+ DC were washed twice to remove anycytokine and co-cultured with 5×10⁴ allogeneic naïve CD4+ T cells inround-bottom 96-well culture plates at increasing DC/T cell ratios. Allco-cultures were carried out in triplicate. DC alone and T cells alonewere used as controls. After 5 days, cells were pulsed with 1 μCi³H-Thymidine (Amersham) for 16 hours before harvesting and countingradioactivity.

Stat3 and Stat5 activation assays. Stable Ba/F3 transfectant cells(˜2.5×10⁷ cells) were starved for 4-6 hours, and then stimulated at 10⁶cells/ml for 15 min with either 10 ng/ml of mIL-3 or 30 ng/ml of hTSLP.After stimulation, cells were harvested and incubated for 15 min at 4degrees C. in lysis buffer containing 50 mM Tris-HCL pH 7.5, 300 mMNaCl, 2 mM EDTA, 0.875% Brij 97, 0.125% NP40, 10 mg/ml aprotinin, 10mg/ml leupeptin, 1 mM PMSF, 1 mM Na3VO4, and 1 mM NaF. Cell lysates wereclarified by centrifugation at 12,000×g for 15 min, and supernatantswere subjected to 8% SDS-PAGE. Proteins were electrotransferred ontonylon membranes (Immobilon-P, Millipore, Bradford, Mass.) and detectedby Western Blot analysis using rabbit Abs against anti-phospho Stat3 andanti-phospho Stat5 (New England Biolabs) or anti-Stat3 and anti-Stat5(Santa Cruz Biotechnology), followed by mouse anti-rabbit Ig HRP.Immunoreactive bands were visualized with enhanced chemiluminescence(ECL) (SuperSignal West Dura Extended Duration Substrate, Pierce,Rockford, Ill.) on ECL film (Kodak). For reprobing, blots were strippedwith 200 mM glycine, 1% SDS, pH 2.5 for 30 min at 65 degrees C.

VI.A. IL-B50 signals via IL-7Rα and Rδ2

The cytokine human IL-B50 has as closest homologs human and mouse IL-7and the recently described mouse TSLP (Sims, et al. (2000) J. Expt'lMed. 192:671-680). In mouse, both IL-7 and TSLP function as T- andB-cell growth and differentiation factors. The signaling receptorcomplexes for mouse IL-7 and mouse TSLP consist of two subunits,respectively mouse IL-7Rα and mouse Rγc (common receptor for IL-2, IL-4,IL-7, IL-9, and IL-15) for mouse IL-7, and mouse IL-7Rα and mouseTSLPR(Rδ1) for the mTSLP ligand (Park, et al. (2000) J. Expt'l Med.192:659-670).

In an attempt to identify the signaling receptor complex for humanIL-B50, Ba/F3 cells were co-transfected with expression constructs forhuman IL-7Rα and an orphan human cytokine receptor known as Rδ2, asubunit related to Rγc and mTSLPR(Rδ1), using the methods describedabove. Any functional relationship between the mTSLPR subunit Rδ1 andthe human Rδ2 had been unclear. Co-transfected Ba/F3 cells showed aproliferative response in the presence of hIL-B50, but not with hIL-7 ormedium. Ba/F3 cells transfected with either hIL-7Rα or hRδ2 alone didnot show a proliferative response with hIL-B50. Additionally, nocross-reactivity between mTSLP and the hTSLP receptor complex wasobserved. These findings establish the signaling complex for humanIL-B50 as consisting of hIL-7Rα and hRδ2.

The corresponding activation status of Stat5 and Stat3 was also measuredin the various BaF3 cell populations. Both Stat5 and Stat3 werephosphorylated upon addition of hTSLP, but only when both hTSLPR andhIL-7Rα was present

VI.B. Expression Analysis of IL-B50, hIL-7Rα and hRδ2

In order to identify target cells capable of responding to IL-B50888888,a large panel of cDNA libraries was analyzed for the simultaneousexpression of both hIL-7Rα and hRδ2, using quatitative PCR. Results ofthe expression analysis, conducted as described in materials andmethods, are presented in FIGS. 4A-4E. In particular, expressionanalysis of the two receptor subunits indicated that they wereco-expressed primarily in activated dendritic cells, monocytes, and Tcells (see, FIGS. 4A, 4B, 4C and 4D) indicating that these cell typesrespond to human IL-B50. As shown in FIG. 4E, IL-B50 was expressed invarious tissue types, with high expression in the human lung.

VI.C. Human IL-B50 Induces Chemokine Expression on Freshly IsolatedMonocyte Population and CD11c+ Blood DC

The spectrum of biological activities induced by IL-B50 was investigatedbased on the overlapping expression patterns of IL-B50 receptorcomponents. cDNA was prepared from human monocytes cultured for 24 hrsin the presence of IL-B50 or IL-7, and the expression of 38 humanchemokines and 20 human chemokine receptors were analyzed byquantitative “real time” PCR. Interestingly, IL-B50 (TSLP) and IL-7influenced the expression of distinct sets of chemokines (Table 1), butdid not affect the expression of chemokine receptors. IL-B50 enhancedthe expression of TARC/CCL17, DC-CK1/PARC/CCL18, MDC/CCL22, andMIP3β/CL19. IL-7 also enhanced expression of TARC/CCL17, MDC/CCL22, andMIP3β/CCL19 but in addition, enhanced expression of IL-8/CXCL8,CTAPIII/CXCL7, ENA78/CXCL5, and GROabg/CXCL123 and decreased theexpression levels of IP-10/CXCL10, I-TACK/CXCL11, SDF1/CXCL12, MCP2/CCL8and MCP4/CCL13 (Table 1).

Additionally, the ability of IL-B50 to stimulate DCs to produce mRNAsfor various cytokines and chemokines was compared with that of GM-CSF,IL-7, CD40-ligand (CD40L) and medium alone as a control, as follows.Purified CD11c+ DCs were cultured for 15-17 hours with IL-B50 (15ng/ml), GM-CSF (100 ng/ml), IL-7 (50 ng/ml), CD40-ligand transfectedL-cells (1 L-cell/4 DC) or medium alone. Total RNA was extracted andstudied using real time quantitative PCR as described above. As shown inFIGS. 14A and 14C, IL-B50 did not stimulate human DCs to produce mRNAfor IL-1α, IL-β, IL-6, IL-12p40, TNF-α, MCP-1, MCP-4, Rantes and MIG,but did stimulate human DCs to produde mRNA for the chemokines TARC, MDCand MIP3-β (FIG. 14B).

The induction of TARC protein by IL-B50 on monocyte and dendritic cellpopulations was confirmed by ELISA. The level of TARC production byCD11c+ dendritic cells was at least ten-fold higher than that ofmonocytes (FIG. 5).

The induction of IL12p75 protein by IL-B50 was also examined. To do so,purified CD11c+ DCs were culture for 24 hours with IL-B50 (15 ng/ml),IL-7 (50 ng/ml), CD40-ligand-transfected L-cells (1 L-cell/4 DC),bacterial lipopolysaccharide (LPS: 1 mg/ml) or medium alone. Culturesupernatant was harvested and bioactive IL12p75 was measured using ahigh-sensitivity ELISA kit. As shown in FIG. 15, IL-B50 did notstimulate human DCs to produce IL12p75 protein.

TABLE 1 Effects of IL-B50 and IL-7 on Chemokine Expression* ChemokineMedia IL-B50 IL-7 CCL1 40.0 1.0 1.4 CCL2 24.8 1.0 1.5 CCL3 31.5 0.7 2.0CCL4 28.9 1.0 1.6 CCL5 30.4 1.1 0.6 CCL7 31.3 0.7 1.4 CCL8 30.2 0.8 0.2CCL11 40.0 1.6 1.4 CCL13 37.3 1.6 0.1 CCL14 40.0 1.3 1.3 CCL15 40.0 1.11.3 CCL16 40.0 2.2 7.0 CCL17 39.8 195.4 20.1 CCL18 35.8 2.9 1.7 CCL1936.7 8.5 8.3 CCL20 40.0 1.2 1.2 CCL21 40.0 1.0 1.0 CCL22 34.3 8.8 3.0CCL24 29.3 2.0 1.2 CCL25 40.0 1.1 1.1 CCL26 38.9 1.1 2.5 CCL27 40.0 0.81.2 CCL28 40.0 1.0 1.0 CXCL1-3 28.7 1.0 4.4 CXCL4 27.9 1.2 1.9 CXCL528.7 1.1 8.0 CXCL6 40.0 1.5 1.3 CXCL7 28.7 1.1 2.0 CXCL8 27.3 1.4 8.5CXCL9 34.9 0.6 0.7 CXCL10 29.7 0.6 0.1 CXCL11 32.9 0.7 0.0 CXCL12 33.10.6 0.2 CXCL13 35.4 0.2 0.8 CXCL14 39.7 1.0 1.0 XCL1 40.0 0.9 3.8 CX3CL140.0 1.4 1.3 *Human monocytes were cultured in the absence or presenceof IL-B50 (50 ng/ml) or IL-7 (50 ng/ml) for 18 h, and expression ofchemokine genes was determined by quantitative PCR. Results areexpressed as 1) C_(T) values of nonactivated samples and 2) folddifference relative to the calibrator sample (media).

VI.D. IL-B50 Activates CD11c+ Dendritic Cells.

Freshly purified immature CD11c+ blood DC are known to spontaneouslymature in culture. As shown in FIG. 6A, loose and irregular clumps inthe DC culture were evident after 24 hrs in medium alone. In thepresence of IL-B50, this maturation process was dramatically enhanced.DC in culture formed tight and round clumps with fine dendrites visibleat the periphery of each clump (FIG. 6B). The IL-B50-induced maturationwas confirmed by analyzing the surface phenotype of DC using flowcytometry. Whereas IL-B50 slightly upregulated the expression of HLA-DRand CD86, it strongly induced the costimulatory molecules CD40 and CD80(FIG. 7). This maturation process was accompanied by an increasedviability of the DC. Additionally, IL-B50 was more potent thanCD40-ligand (CD40L) and IL-7 in upregulating CD40 and CD80 (FIGS.8A-8C). A titration of IL-B50 using log dilutions of the cytokine showedthat both the effect on survival and the induction of costimulatorymolecules on DC was maximal at 15 ng/ml and above, and still significantat concentrations as low as 15 pg/ml.

The T cell stimulatory capacity of CD11+ DC, cultured 24 hrs in mediumalone or in the presence of IL-B50, was analyzed. DCs were coculturedwith 5×10 ⁴ naïve CD4+ CD45RA+ allogeneic T cells at increasing DC/Tcell ratios. As assessed by ³H-thymidine incorporation at day 5 of thecoculture, DC cultured with IL-B50 induced up to 10-fold stronger naïveT cell proliferation as compared to DC cultured in medium (FIG. 9).

Additionally, the T cell stimulatory capacity of CD11+ DC, cultured withLPS, medium alone, IL-7, CD40-ligand and IL-B50, was compared bydetermining ³H-thymidine incorporation. As shown in FIG. 10,IL-B50-activated DCs were more potent than DCs activated withCD40-ligand, IL-7 and LPS in inducing proliferation of allogeneic naïveCD4 T cells.

DCs, cultured in medium alone, IL-B50, CD40-ligand, IL-7 and LPS, werecocultured with naïve CD4 T cells. After 6 days of coculture, CD4 Tcells were restimulated for 24 hours with anti-CD3 and anti-CD28 and theculture supernatants were analyzed by ELISA to quantify the cytokineproduction by T cells. As shown in FIGS. 11A-11E, IL-B50-activated humanDCs primed naïve CD4 T cells to produce IL-4, IL-13 and TNF-α, butinhibited production of IL-10 and IFN-γ.

DCs were cultured for 24 hours in medium alone, IL-B50, IL-7,CD40-ligand or LPS, to prime purified naïve CD8 T cells over 6 days incoculture. IL-B50-activated DCs strongly induced expansion of allogeneicnaïve CD8 T cells (FIG. 12), as well as the expression of perforin (FIG.13).

The data herein indicate, among other things, that human IL-B50 is anovel hematopoietic cytokine most closely related to IL-7. It representsa human ortholog of mTSLP. The human IL-B50 signaling makes use of thecombination of hIL-7Rα and hRδ2, which together form a novelhematopoietic cytokine receptor. Both receptor subunits are notablypresent on macrophages and dendritic cells, indicating functionaleffects of the cytokine on those cell types, and mediating functionsprovided by those cell types. The human IL-B50 also promotes thephosphorylation of STAT3 and STAT5 transcription factors.

Therapeutic uses of IL-B50 are apparent. For example, SCID patients withmutations in IL-7Rα are T-cell deficient. Since IL-B50 uses IL-7Rα,IL-B50 share signaling pathway components, and may play a significantrole in human T-cell differentiation and may enhance T-cell recovery incircumstances of T-cell depletion.

Likewise, IL-B50 antagonists are useful. The antagonists take variousforms such as ligand muteins, antibodies to ligand, and antibodies toreceptor, e.g., which block ligand binding. Since the hIL-B50 likelyplays a role in the development of T- and B-cell lymphomas, thenblocking IL-B50 signaling, either at the ligand or at its receptorcomponents, is useful in treatment of some of these lymphomas.

Herein, based upon the binding studies, hIL-B50 receptor subunit mappingidentified IL-7Rα and novel human receptor Rδ2 (a close relative ofhuman γc or IL-2Rγ) as signaling receptor complex (co-expression inBa/F3 cells delivers a proliferative signal in response to IL-B50).Receptor expression profiles indicate both IL-7Rα and Rδ2 are primarilyexpressed on dendritic cells, though they are both also expressed inmonocytes. The dendritic cell expression indicates a role for thecytokine in maturation of cells or pathways important in antigenpresentation, indicating use of the cytokine for expansion, e.g., exvivo, of antigen presenting cells.

All citations herein are incorporated herein by reference to the sameextent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referenceincluding all figures and drawings.

Many modifications and variations of this invention, as will be apparentto one of ordinary skill in the art can be made to adapt to a particularsituation, material, composition of matter, process, process step orsteps, to preserve the objective, spirit and scope of the invention. Allsuch modifications are intended to be within the scope of the claimsappended hereto without departing from the spirit and scope of theinvention. The specific embodiments described herein are offered by wayof example only, and the invention is to be limited by the terms of theappended claims, along with the full scope of equivalents to which suchclaims are entitled; and the invention is not to be limited by thespecific embodiments that have been presented herein by way of example.

1. An isolated or purified antibody or an antigen binding fragmentthereof which binds with selectivity to a human IL-B50 receptor complexcomprising a human IL7Rα subunit comprising amino acids 21 to 459 of SEQID NO:2, and a human Rδ2 subunit comprising amino acids 23 to 371 of SEQID NO:4, wherein the antibody does not bind to the polypeptide encodedby SEQ ID NO:2 or to the polypeptide encoded by SEQ ID NO:4.
 2. Theantibody of claim 1, wherein the antibody is a monoclonal antibody or anantigen binding fragment thereof.
 3. The antibody of claim 1, whereinthe antibody is a humanized monoclonal antibody or an antigen bindingfragment thereof.
 4. The antibody of claim 1, wherein the antibody is ahuman antibody or an antigen binding fragment thereof.
 5. The antibodyof claim 1, which inhibits signaling of IL-B50 through the receptorcomplex.
 6. The antibody of claim 5, wherein the antibody is amonoclonal antibody or an antigen binding fragment thereof.
 7. Theantibody of claim 5, wherein the antibody is a humanized monoclonalantibody or an antigen binding fragment thereof.
 8. The antibody ofclaim 5, wherein the antibody is a human monoclonal antibody or anantigen binding fragment thereof.
 9. A pharmaceutical formulationcomprising the antibody of claim 1 or an antigen binding fragmentthereof and one or more pharmaceutically acceptable carriers.